Methods of diagnosing a disease and methods of monitoring treatment of a disease by quantifying a non-reducing end glycan residual compound and comparing to a second biomarker

ABSTRACT

Provided herein are methods of diagnosing or monitoring the treatment of abnormal glycan accumulation or a disorder associated with abnormal glycan accumulation.

CROSS-REFERENCE

This application is a continuation application of U.S. application Ser.No. 15/097,218, filed Apr. 12, 2016, which is a continuation of U.S.application Ser. No. 14/462,285, filed Aug. 18, 2014, now U.S. Pat. No.9,340,822, issued on May 17, 2016, which is a continuation of U.S.application Ser. No. 13/629,321, filed Sep. 27, 2012, now U.S. Pat. No.8,809,009, issued on Aug. 19, 2014, which claims the benefit of U.S.Provisional Application No. 61/561,698, filed Nov. 18, 2011, and is acontinuation-in-part of U.S. application Ser. No. 13/550,106, filed Jul.16, 2012, now U.S. Pat. No. 8,771,974, issued on Jul. 8, 2014, which isa continuation of U.S. application Ser. No. 12/649,110, filed Dec. 29,2009, now U.S. Pat. No. 8,232,073, issued on Jul. 31, 2012, which claimsthe benefit of U.S. Provisional Application No. 61/238,079, filed Aug.28, 2009, U.S. Provisional Application No. 61/164,365, filed Mar. 27,2009, and U.S. Provisional Application No. 61/142,291, filed Jan. 2,2009; each of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

Many human diseases are caused by or correlated with changes inglycosylation. In order to use these changes as biomarkers of disease,analytical methods are used to quantify the changes. The publishedmethods use antibodies, chromatography and/or mass spectrometrytechniques to resolve and quantify the intact or partially intactglycans. These methods are challenging due to the complexity and numberof possible glycan structures present in biological samples. In a singledisease state there can be thousands of different novel glycanstructures that are present; however, each on their own is a weak markerof disease.

SUMMARY OF THE INVENTION

Described herein are populations of glycans that are transformed intopopulations of biomarkers using glycan degradation enzymes. In someembodiments, described herein are populations of glycosaminoglycans thatare transformed into populations of oligosaccharides usingglycosaminoglycan lyases. Further described herein are the use ofanalytical instruments to characterize the population of biomakers(i.e., non-reducing end glycan residual compounds, such asmonosaccharides) in order to provide relevant information about thepopulation of biomarkers, the population of biomarkers and thebiological sample that provided the population of biomarkers. In someembodiments, described herein are the use of analytical instruments tocharacterize the population of oligosaccharides in order to providerelevant information about the population of oligosaccharides, thepopulation of glycosaminoglycans and the biological sample that providedthe population of glycosaminoglycans.

Provided in certain embodiments herein are methods of detecting glycanaccumulation and/or abnormal glycan biosynthesis and/or degradation in abiological sample, the method comprising:

-   -   a. transforming a glycan of a biological sample with a glycan        degradation enzyme to liberate a glycan residual compound from        the non-reducing end of the glycan;    -   b. measuring the amount of the glycan residual compound        liberated by the functioning glycan degradation enzyme with an        analytical device.

In some embodiments, a method described herein comprises a method ofdiagnosing an individual as having a disease or condition associatedwith abnormal glycan biosynthesis, degradation, or accumulation, themethod comprising:

-   -   a. generating a biomarker comprising of one or more non-reducing        end glycan residual compound, wherein the biomarker is generated        by treating a population of glycans, in or isolated from a        biological sample from the individual, with at least one        digesting glycan enzymes, wherein prior to enzyme treatment, the        biomarker is not present in abundance in samples from        individuals with the disease or condition relative to        individuals without the disease or condition, and    -   b. using an analytical instrument to detect the presence of        and/or measure the amount of the biomarker produced and        displaying or recording the presence of or a measure of a        population of the biomarker.

In some embodiments, the presence of and/or measure the amount of thebiomarker is utilized to determine the presence, identity, and/orseverity of the disease or condition.

Provided in certain embodiments herein is a method of diagnosing anindividual as having a disease or condition associated with abnormalglycan biosynthesis, degradation, or accumulation, the methodcomprising:

-   -   a. transforming a glycan of a biological sample with a glycan        degradation enzyme to liberate a glycan residual compound from        the non-reducing end of the glycan;    -   b. measuring the amount of the glycan residual compound        liberated by the functioning glycan degradation enzyme with an        analytical device; and    -   c. determining whether the amount of liberated glycan residue is        abnormal.

In some embodiments, provided herein is a method of monitoring thetreatment of a disorder associated with the abnormal degradation,biosynthesis and/or accumulation of glycans, the method comprising:

-   -   a. following administration of an agent for treating a disorder        associated with the abnormal degradation, biosynthesis and/or        accumulation of glycans to an individual in need thereof, using        an analytical instrument to measure the amount of a population        of a biomarker comprising a non-reducing end glycan residual        compounds present in a transformed biological sample, the        biomarker being generated by treating a population of glycans,        in or isolated from a biological sample from the individual,        with at least one digesting glycan enzyme(s), wherein prior to        enzyme treatment, the biomarker is not present in abundance in        samples from individuals with the disease or condition relative        to individuals without the disease or condition, and    -   b. determining whether or not the amount of biomarker has        decreased or increased at a slower rate compared to the amount        or rate of increase prior to administration of the agent for        treating a disorder associated with the abnormal degradation,        biosynthesis and/or accumulation of glycans.

In some embodiments, the disorder associated with the abnormaldegradation, biosynthesis and/or accumulation of glycans is a lysosomalstorage disease, a cancerous disease, an inflammatory disease, aninfectious disease, a central nervous system disease, or acardiovascular disease. In some embodiments, the normally functioningglycan degradation enzyme is a glycosidase, sulfatase, phosphorylase,deacetylase, or a combination thereof. In some embodiments, the normallyfunctioning glycan degradation enzyme is a glycosidaseis selected froman exo-glycosidase and an endo-glycosidase. In some embodiments, theglycan residual compound is a monosaccharide, sulfate, phosphate,acetate, or a combination thereof.

In some embodiments, transforming a glycan of a biological sample with anormally functioning glycan degradation enzyme comprises transforming aglycan of a biological sample with a plurality of normally functioningglycan degradation enzymes. In some embodiments, the glycan is treatedwith a plurality of normally functioning glycan degradation enzymesconcurrently, sequentially, or a combination thereof. In someembodiments, prior to measuring the amount of a population ofnon-reducing end glycan residual compounds, the non-reducing end glycanresidual compounds are labeled with a detectable label. In someembodiments, the detectable label is a mass label, a radioisotope label,a fluorescent label, a chromophore label, or affinity label. In someembodiments, the amount of liberated glycan is measured using UV-Visspectroscopy, IR spectroscopy, mass spectrometry, or a combinationthereof.

Provided in certain embodiments herein is a method of diagnosing anindividual as having a disease or condition associated with abnormalglycan biosynthesis, degradation, or accumulation, the methodcomprising:

-   -   a. generating a biomarker comprising of one or more non-reducing        end glycan residual compound, wherein the biomarker is generated        by treating a population of glycans, in or isolated from a        biological sample from the individual, with at least one        digesting glycan enzymes, wherein prior to enzyme treatment, the        biomarker is not present in abundance in samples from        individuals with the disease or condition relative to        individuals without the disease or condition, and    -   b. using an analytical instrument to detect the presence of        and/or measure the amount of the biomarker produced and        displaying or recording the presence of or a measure of a        population of the biomarker;

wherein the presence of and/or measure the amount of the biomarker isutilized to determine the presence, identity, and/or severity of thedisease or condition.

In some embodiments, the disease or disorder is caused by an abnormallyfunctioning glycan degradation enzyme and wherein the abnormallyfunctioning glycan degradation enzyme and the normally functioningglycan degradation enzyme are of the same type. In some embodiments, theabnormally functioning glycan degradation enzyme functions abnormally asa result of being present in an abnormally low amount, functioningimproperly, or a combination thereof. In some embodiments, the abnormalglycan accumulation comprises the accumulation of abnormal amounts ofglycans. In some embodiments, the abnormal glycan accumulation comprisesthe accumulation of abnormal amounts of normal glycans. In someembodiments, the abnormal glycan accumulation comprises the accumulationof abnormal amounts of abnormal glycans.

In some embodiments, the normally functioning glycan degradation enzymeis a glycosidase, sulfatase, phosphorylase, deacetylase, or acombination thereof. In some embodiments, the normally functioningglycan degradation enzyme is a glycosidase selected from anexo-glycosidase and an endo-glycosidase. In some embodiments, theglycosidase is an exo-glycosidase selected from the group consisting ofa galactosidase, and a glucuronidase.

In some embodiments, the glycan residual compound is a monosaccharide.In some embodiments, the glycan residual compound is sulfate, phosphate,acetate, or a combination thereof. In some embodiments, a biologicalsample is purified prior to transforming a glycan thereof. In someembodiments, the process of purifying a biological sample comprisesremoving monosaccharides therefrom, removing sulfates therefrom,removing phosphates therefrom, removing acetate therefrom, or acombination thereof. In some embodiments, transforming a glycan of abiological sample with a normally functioning glycan degradation enzymecomprises transforming a glycan of a biological sample with a pluralityof normally functioning glycan degradation enzymes. In some embodiments,the glycan is treated with a plurality of normally functioning glycandegradation enzymes concurrently, sequentially, or a combinationthereof.

In some embodiments, the disorder associated with an abnormal glycanaccumulation is MPS I, MPS II, MPS IIIA, MPS IVA, MPSVI, or FabryDisease. In some embodiments, determining whether the amount ofliberated glycan residue is abnormal comprises labeling the glycanresidue with a detectable label and measuring the amount of labeledglycan residue with an analytical instrument. In some embodiments, thedetectable label is a mass label, a radioisotope label, a fluorescentlabel, a chromophore label, or affinity label. In some embodiments, theamount of liberated glycan is measured using UV-Vis spectroscopy, IRspectroscopy, mass spectrometry, or a combination thereof.

Provided herein, in certain embodiments, is a method of diagnosing anindividual as having a disease or condition (e.g., associated withabnormal glycan biosynthesis, degradation, or accumulation), the methodcomprising:

-   -   a. generating a first biomarker comprising a glycan residual        compound, wherein the first biomarker is generated by treating a        population of glycans, in or isolated from a biological sample        from the individual, with at least one digesting glycan enzyme,        wherein prior to enzyme treatment, the first biomarker is not        present in abundance in samples from individuals with the        disease or condition relative to individuals without the disease        or condition,    -   b. generating a second biomarker comprising a glycan residual        compound, wherein the second biomarker is generated by treating        a population of glycans, in or isolated from a biological sample        from the individual, with at least one digesting glycan enzyme        in the same or different digestion step as provided in step (a),        wherein prior to enzyme treatment, the second biomarker is not        present in abundance in samples from individuals with the        disease or condition relative to individuals without the disease        or condition,    -   c. using an analytical instrument to detect the presence of        and/or measure the amount of the first and second biomarker        produced and displaying or recording the presence of or a        measure of a population of the first and second biomarkers, and    -   d. monitoring and/or comparing the amounts of the first and        second biomarkers in a biological sample;        wherein the presence of and/or measure of the amounts of the        first and second biomarkers are utilized to determine the        presence, identity, and/or severity of the disease or condition.

In some embodiments, the first biomarker is a non-reducing end glycanresidual compound. In some embodiments, the disease or disorder iscaused by an abnormally functioning glycan degradation enzyme andwherein the abnormally functioning glycan degradation enzyme and thedigesting glycan enzyme are of the same type. In some embodiments, thenon-reducing end glycan residual compound is a monosaccharide. In someembodiments, the non-reducing end glycan residual compound is not amonosaccharide.

In some embodiments, the second biomarker is derived or generated fromthe reducing end of the same glycan from which the first non-reducingend glycan residual compound biomarker was generated. In someembodiments, the second biomarker is derived or generated from theinternal oligosaccharide structures of the same glycan from which thefirst non-reducing end glycan residual compound biomarker was generated.In some embodiments, the disease or disorder is caused by the abnormalfunction of a glycan degradation enzyme in the individual, and whereinthe second biomarker can be generated by treating the first non-reducingend glycan residual compound biomarker with the glycan degradationenzyme that is functioning abnormally in the individual.

In some embodiments, the disease or condition associated with abnormalglycan biosynthesis, degradation, or accumulation is a lysosomal storagedisease. In some embodiments, the lysosomal storage disease isMucopolysaccharidosis. In some embodiments, the Mucopolysaccharidosis isMPS I, II, IIIA, IIIB, IIIC, IIID, IVA, IVB, VI, or VII. In someembodiments, the disease or condition associated with abnormal glycanbiosynthesis, degradation, or accumulation is MetachromaticLeukodystrophy or Krabbe disease. In some embodiments, the disease orcondition associated with abnormal glycan biosynthesis, degradation, oraccumulation is Gangliosidosis. In some embodiments, the Gangliosidosisis Tay Sachs, Sandhoff, AB Variant, or GM-1 Gangliosidoses.

In some embodiments, the presence of and/or measure of the firstnon-reducing end glycan residual compound biomarker in combination withor in relation to the second biomarker is utilized to monitor thetreatment of a disorder associated with the abnormal biosynthesis ofglycans. In some embodiments, the presence of and/or measure of thefirst non-reducing end glycan residual compound biomarker in combinationwith or in relation to the second biomarker is utilized to monitor thetreatment of a disorder associated with the abnormal degradation oraccumulation of glycans. In some embodiments, the treatment is enzymereplacement therapy. In some embodiments, the absence of an increase inthe second biomarker combined with a reduction in the non-reducing endglycan residual compound biomarker indicates a positive response totreatment of the disorder associated with abnormal degradation oraccumulation of glycans.

In some instances, a method described herein comprises utilization of afirst biomarker that is a non-reducing end glycan biomarker, e.g., asset forth in US 2010/0184013, which is incorporated by reference hereinin its entirety. In certain embodiments, the first biomarker is a C4-C5non-reducing end saturated oligosaccharide. In some embodiments, thenon-reducing end residue of the first biomarker (e.g., one or moredisaccharide and/or one or more trisaccharide) are free of carbon-carbonunsaturation.

In some embodiments, the abnormal glycan accumulation or disorderassociated therewith is caused by an abnormally functioning glycandegradation enzyme and wherein the abnormally functioning glycandegradation enzyme and glycan degradation enzyme are of the same type(e.g., the glycan degradation utilized in the transformation process isa functioning glycan degradation enzyme whereas the abnormallyfunctioning enzyme is not, such as due to deletions, insertions,substitutions, or other modifications to the enzyme sequence). Incertain embodiments, the abnormally functioning glycan degradationenzyme functions abnormally as a result of being present in anabnormally low amount, functioning improperly, or a combination thereof.In some embodiments, the abnormal glycan accumulation comprises theaccumulation of abnormal amounts of glycans. In certain embodiments, theabnormal glycan accumulation comprises the accumulation of abnormalamounts of normal glycans. In some embodiments, the abnormal glycanaccumulation comprises the accumulation of abnormal amounts of abnormalglycans.

In certain embodiments, the biomarker is not present in the originalbiological sample. In some embodiments, the biomarker is not present inthe biological sample after isolating a population of glycans therefrom(e.g., prior to transformation of the glycan according to a processdescribed herein).

In certain embodiments, the normally functioning glycan degradationenzyme is a glycosidase, sulfatase, phosphorylase, deacetylase or acombination thereof. In some embodiments, the normally functioningglycan degradation enzyme is a glycosidase selected from anexo-glycosidase and an endo-glycosidase. In certain embodiments, theglycosidase is an exo-glycosidase selected from the group consisting ofa galactosidase, and a glucuronidase. In some embodiments, the generatedbiomarker is a glycan residual compound. In some embodiments, the glycanresidual compound is a monosaccharide. In certain embodiments, theglycan residual compound is sulfate, phosphate, acetate, or acombination thereof. In certain embodiments, the glycan residualcompound has a molecular weight of less than 2000 g/mol, less than 1500g/mol, less than 1000 g/mol, less than 500 g/mol, less than 400 g/mol,less than 300 g/mol, less than 260 g/mol, less than 200 g/mol, less than100 g/mol, or the like (e.g., prior to tagging with any detectable labelthat may be included in a process described herein).

In some embodiments, any process described herein further comprisespurifying a biological sample prior to transforming a glycan thereof. Insome embodiments, the process of purifying a biological sample comprisesremoving monosaccharides therefrom, removing sulfates therefrom,removing phosphates therefrom, removing acetate therefrom, or acombination thereof.

In certain embodiments, transforming a glycan of a biological samplewith a normally functioning glycan degradation enzyme comprisestransforming a glycan of a biological sample with a plurality ofnormally functioning glycan degradation enzymes. In some embodiments,the glycan is treated with a plurality of normally functioning glycandegradation enzymes concurrently, sequentially, or a combinationthereof.

In specific embodiments, a disorder associated with an abnormal glycanaccumulation is any disorder described in Tables 1-4 (e.g., MPS I) andthe normally functioning glycan degradation enzyme is any enzymedescribed in Tables 1-4 (e.g., L-iduronidase).

In some embodiments, determining whether the amount of liberated glycanresidue is abnormal comprises labeling the glycan residue with adetectable label and measuring the amount of labeled glycan residue withan analytical instrument. In certain embodiments, the detectable labelis a mass label, a radioisotope label, a fluorescent label, achromophore label, or affinity label. In some embodiments, the amount ofliberated glycan is measured using UV-V is spectroscopy, IRspectroscopy, mass spectrometry, or a combination thereof.

Provided in some embodiments herein is a method of monitoring thetreatment of a disorder associated with the abnormal degradation,biosynthesis and/or accumulation of glycans, the methods comprising:

-   -   a. following administration of an agent for treating a disorder        associated with the abnormal degradation, biosynthesis and/or        accumulation of glycans to an individual in need thereof, using        an analytical instrument to measure the amount of a population        of a non-reducing end glycan residual compounds present in a        transformed biological sample that has been prepared by:        -   treating a population of glycans, in or isolated from a            biological sample taken from the individual, with at least            one normally functioning glycan degradation enzyme to            liberate non-reducing end glycan residual compound;    -   b. determining whether or not the amount of liberated        non-reducing end glycan residue has decreased or increased at a        slower rate compared to the amount or rate of increase prior to        administration of the agent for treating a disorder associated        with the abnormal degradation, biosynthesis and/or accumulation        of glycans.

In some embodiments, the disorder associated with the abnormaldegradation, biosynthesis and/or accumulation of glycans is a lysosomalstorage disease, a cancerous disease, or an infectious disease. Incertain embodiments, the normally functioning glycan degradation enzymeis a glycosidase, sulfatase, phosphorylase, deacetylase, or acombination thereof. In some embodiments, the normally functioningglycan degradation enzyme is a glycosidase selected from anexo-glycosidase and an endo-glycosidase. In certain embodiments, theglycan residual compound is a monosaccharide, sulfate, phosphate,acetate, or a combination thereof. In some embodiments, transforming aglycan of a biological sample with a normally functioning glycandegradation enzyme comprises transforming a glycan of a biologicalsample with a plurality of normally functioning glycan degradationenzymes. In certain embodiments, the glycan is treated with a pluralityof normally functioning glycan degradation enzymes concurrently,sequentially, or a combination thereof.

In some embodiments, prior to measuring the amount of a population ofnon-reducing end glycan residual compounds, the non-reducing end glycanresidual compounds are labeled with a detectable label. In certainembodiments, the detectable label is a mass label, a radioisotope label,a fluorescent label, a chromophore label, or affinity label. In someembodiments, the amount of liberated glycan is measured using UV-Visspectroscopy, IR spectroscopy, mass spectrometry, or a combinationthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates compounds present in a normal biological sample notsubject to an enzymatic glycan residual liberation process describedherein.

FIG. 2 illustrates compounds present in a normal biological subject toan enzymatic glycan residual liberation process described herein.

FIG. 3 illustrates compounds present in a biological sample of anindividual suffering from a disorder associated with abnormal glycanaccumulation not subject to an enzymatic glycan residual liberationprocess described herein.

FIG. 4 illustrates compounds present in a biological sample of anindividual suffering from a disorder associated with abnormal glycanaccumulation subject to an enzymatic glycan residual liberation processdescribed herein.

FIG. 5 illustrates a scheme for determining non-reducing ends andinternal disaccharide. Eliminative depolymerization of a heparan sulfateoligosaccharide with heparan lyase results in the release of internaldisaccharide residues (dashed arrows) that contain an unsaturated uronicacid moiety (dotted circles). Because of its terminal location, thenon-reducing end liberated from the left end of the chain as drawn lacksthe Δ4,5-double bond and is 18 amu larger than a corresponding internaldisaccharide. Reductive amination with aniline ([12C₆]An) facilitatesseparation of the various disaccharides by LC/MS, yielding the m/zvalues for the molecular ions indicated within the parentheses. Theglycan structures are graphically represented by geometric symbols,which are defined in the lower part of the FIG. 43. To simplify therepresentation of constituent oligosaccharides from glycosaminoglycans,we use a Disaccharide Structure Code (DSC)15. In DSC, a uronic acid isdesignated as U, G, I, or D for an unspecified hexuronic acid,D-glucuronic acid, L-iduronic acid, or Δ4,5-unsaturated uronic acid,respectively. The hexosamines are designated in upper case forglucosamine and lower case for galactosamine, and the N-substituent iseither H, A, S or R for hydrogen, acetate, sulfate, or some othersubstituent, respectively. The presence and location of ester linkedsulfate groups are depicted by the number of the carbon atom on whichthe sulfate group is located or by 0 if absent. For example, I2S6 refersto a disaccharide composed of 2-sulfoiduronicacid-N-sulfoglucosamine-6-sulfate, whereas D2S6 refers to samedisaccharide, but bearing a Δ4,5-double bond in the uronic acid.

FIG. 6 illustrates MPS Non-reducing end carbohydrates. The defectiveenzyme for each MPS subclass is displayed along with the liberated NREcarbohydrates characteristic of MPS I, II, IIIA, IIIB, IIIC, IIID, VIand VII using geometric symbols. The matrices show all NRE carbohydratesthat are theoretically possible for each MPS subclass using the DSC. Theboxes with a black background and whiteface font depict structures thatwere detected and whose identities were confirmed by theirco-chromatography and identical mass spectra as standards, as well astheir sensitivity to exoglycosidases or propionic acid anhydride.Suspected structures shown in boxes with a gray background are impliedfrom the liquid chromatography/mass spectra data, i.e. their size andcontent of sulfate and acetate groups are consistent with the proposedstructures. The structures in boxes with a white background aretheoretically possible, but have not been observed. The m/z values forboth the free molecular ions and adduction ions formed with the ionpairing reagent dibutylamine (DBA) are listed. The single letterdesignations for the variously modified sugars are described in FIG. 5.The glycan structures are graphically represented by geometric symbols,which are defined in the lower part of the figure

FIG. 7 illustrates analysis of non-reducing ends found in MPS I andSanfilippo heparan sulfate. (a) Depolymerized heparan sulfate from MPS Ifibroblasts (GM01391) was tagged with [12C6]aniline and mixed withstandard [13C6]aniline-labeled unsaturated disaccharides and I0S0. Thesample was analyzed by LC/MS and the extracted ion current for all knownNRE and internal disaccharides was recorded: peak 1, D0A0; peak 2, D0S0;peak 3, D0A6; peak 4, D0S6; peak 5, D2A0; peak 6, D2S0; and peak 7,D2S6. The asterisk marks the [12C6]aniline tagged NRE, which comigratedwith [13C6]aniline-labeled I0S0 standard (inset). (b) Mass spectrum forthe I0S0 peak shown in panel A. GAGs purified from (c) MPS IIIA(GM00643), (d) MPS IIIB (GM01426), (e) MPS IIIC (GM05157) and (f) MPSIIID (GM17495) fibroblasts were subjected to NRE analysis. Forsimplicity, only the extracted ion current for m/z values correspondingto monosaccharide and trisaccharide (dp3) NREs are shown for eachsample. When the NRE structure was identified by comparison withcommercially available standards, the name is indicated in DSC andglycan symbols. The dp3(0Ac,4S) NRE residues in the MPS IIIA and the MPSIIIC samples were detected as adduction ions with the ion pairingreagent ([M−2H+DBA]−1); hence their m/z values were increased by 129 amu(see FIG. 6). The insets in panels c and f show the mass spectra for themonosaccharide biomarkers S0 and H6, respectively, and the corresponding[13C6]aniline tagged standards (arrows in panels c and f).

FIG. 8 illustrates systematic diagnostic screening of GAG samples forvarious MPS disorders. Shown is a flow chart for MPS discovery based onthe detection of diagnostic non-reducing end glycans present in GAGsamples extracted from patient or animal model sources. The detectioncriteria are based on NRE size (monosaccharide, disaccharide andtrisaccharide), m/z value, and structural features (number of acetates(Ac) or sulfates (S)). For a complete unknown, portions of the sampleare analyzed in parallel for heparan sulfate and chondroitin/dermatansulfate NREs.

FIG. 9 illustrates a comparison of total heparan sulfate toN-sulfoglucosamine (S0) in MPS IIIA samples. (a) Heparan sulfate fromnormal (black bars) and MPS IIIA (light grey bars) urine was analyzed.The individual disaccharides and NRE N-sulfoglucosamine (S0) wasquantitated relative to standards. Since trisaccharide standards are notavailable, the values of the extracted ion current for S0U2S0 are shown.(b) Analysis of normal (black bars) and MPS IIIA (light grey bars) brainheparan sulfate as in panel A. (c) MPS IIIA cells underwent enzymereplacement by incubation with 0.06 mU/ml of sulfamidase for 48 hoursprior to GAG extraction and subsequent analysis. The amount of heparansulfate (black bars), the monosaccharide biomarker S0 (light grey bars),and the trisaccharide biomarker S0U2S0 (dark grey bars) were measuredand compared to samples from cells without enzyme supplementation. Thebars represent the average±standard deviation, n=3.

FIG. 10 illustrates a structural comparison of MPS I NRE biomarker withI0S0 standard. Tandem mass spectrometry was carried out on[¹³C₆]aniline-tagged I0S0 standard. The predominant daughter ions andtheir assignments based on m/z values are indicated in the mass spectrum(a). A schematic representation of the primary inter-ring cleavage ofI0S0 is also shown. The m/z values are shown next to each fragment ion.After labeling with [¹²C₆]aniline, the putative I0S0 NRE found inheparan sulfate from cultured fibroblasts from a MPS I patient (GM01391)was also subjected to tandem mass spectrometry (b). The m/z values shownin each CID spectrum are consistent with the difference in mass between[¹³C₆]aniline and [¹²C₆]aniline. Identity between the standard and theMPS I NRE is indicated by the similar CID spectra.

FIG. 11 illustrates analysis of non-reducing end structures found in MPSI, MPS II and MPS VII. GAG samples purified from (a,d) MPS I (GM01391),(b,e) MPS II (GM00615) and (c,f) MPS VII (GM02784) fibroblasts weresubjected to enzymatic depolymerization with heparan lyase (a,b,c) orchondroitinase ABC (d,e,f) followed by GRIL-LC/MS analysis. Theaccumulative extracted ion current for m/z values corresponding toheparan sulfate and chondroitin/dermatan sulfate NRE structures detectedin each sample is shown. In the absence of authentic standards, therelative abundance of the individual biomarkers cannot be derived fromthese spectra due to differences in ionization efficiencies. Theidentification of the uronic acids is based on chromatographicseparation of isobaric species (e.g. G0Sa0 and G0S6 resolve from I0S0and I0S6, respectively), differential sensitivity to α-L-iduronidase,and inference based on the nature of the enzyme deficiency in the cells.Putative structures are indicated by both DSC and glycan symbols as wellas their m/z values. In cases where two isobaric species could not bediscriminated by CID analysis (I0a4/I0a6 in panel d and I2a4/I2a6 inpanel e), both species are shown.

FIG. 12 illustrates removal of the non-reducing end biomarker in MPS Iby α-iduronidase treatment. An equal amount of heparan sulfate isolatedfrom fibroblasts of a patient with MPS I (GM01391) was treated withrecombinant α-iduronidase or with BSA. The samples were then tagged with[¹²C₆] aniline, mixed with 10 pmole of [¹³C₆]aniline-labeled I0S0standard, and analyzed by GRIL-LC/MS. The mass spectrum for theBSA-treated samples shows the MPS I NRE and the I0S0 standard (a). Themass spectrum of the iduronidase-treated sample shows the loss of theMPS I biomarker (b). The m/z values for all species detected areindicated above the major peaks.

FIG. 13 illustrates removal of the non-reducing end biomarker found inMPS II by Iduronate-2-sulfatase. Equal amounts of heparan sulfateisolated from fibroblasts from an MPS II patient (GM00615) were treatedwith either BSA or recombinant iduronate-2-sulfatase prior to NREanalysis. The accumulative extracted ion current for m/z valuescorresponding to 2-O-sulfated and non-2-O-sulfated NRE species for theBSA-treated sample (black trace) and the iduronate-2-sulfatase-treatedsample (red trace) are shown in the chromatogram. The NRE structuresconsistent with the m/z values for the species detected after eachtreatment are shown.

FIG. 14 illustrates GRIL-LC/MS analysis of MPS VI chondroitin sulfateNRE. GAG purified from MPS VI (GM00519) fibroblasts was enzymaticallydepolymerized with chondroitinase ABC followed by GRIL-LC/MS analysis.The accumulative extracted ion current for m/z values corresponding toNRE monosaccharide structures GaINAc6S (a6) and GaINAc4S (a4) standardslabeled with [¹³C₆] aniline are shown (bottom red trace) and theendogenous a4 labeled with [¹²C₆] aniline detected in the MPS VI sampleis shown (top black trace). The mass spectrum are indicated for the a4peak in the top trace is shown in the inset with the m/z values for theisotopic clusters for the endogenous [¹²C₆]aniline-tagged a4(m/z=377.11) and the [¹³C₆]aniline-labeled a4 standard (m/z=383.13).

FIG. 15 illustrates removal of the non-reducing end biomarker in MPSIIIA by sulfamidase. Heparan sulfate purified from fibroblasts from twoMPS IIIA patients (GM00643 and GM00934) was treated with either BSA(black bars) or sulfamidase (light grey bars) prior to GRIL-LC/MSanalysis. [¹³C₆]aniline-labeled S0 standard (10 pmol) was added to eachsample and used to calculate the recovery of S0.

FIG. 16 illustrates CID analysis of NRE trisaccharides found inSanfilippo sub-classes. Representative trisaccharide NRE structuresdetected in heparan sulfate from fibroblasts of MPS IIIA, MPS IIIB andMPS IIIC patients were subjected to collision induced dissociation. Themost likely structures are indicated along with the product ionsdetected. The structural parameters for each parent ion are displayedbelow each structure. To confirm the presence of an unsubstitutedglucosamine residue in the MPS IIIC trisaccharides, aniline-labeledsamples were acylated with propionic anhydride (PA), which reacts withboth primary and secondary amines. The solid arrows point out primaryand secondary amines susceptible to acylation by propionic anhydride.All of the MPS IIIC trisaccharides gained mass consistent with theaddition of two propionyl groups. In contrast, the MPS IIIA and IIBtrisaccharides picked up a single propionyl group due to proprionylationat the bridging secondary amine derived from reductive amination withaniline. Thus, the addition of a second proprionate group to MPS IIICtrisaccharides is consistent with their containing an unsubstitutedglucosamine unit. Due to the detection of only two product ions for theMPS IIIC NRE trisaccharide, two potential structures are possible.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, such embodiments are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention. It isintended that the following claims define the scope of the invention andthat methods and structures within the scope of these claims and theirequivalents be covered thereby.

Glycosaminoglycans comprise a reducing end and a non-reducing end.Normal biological processes degrade glycosaminoglycans (such as heparansulfate which has a normal component of about 50-80 kDa) intomonosaccharides. Disorders associated with abnormal glycosaminoglycandegradation, biosynthesis, and/or accumulation can result in anaccumulation of glycosaminoglycans and fragments thereof.

Many human diseases are caused by or correlated with changes inglycosaminoglycans. In order to use these changes as biomarkers ofdisease, analytical methods are used to quantify the changes. Somemethods use antibodies, chromatography and/or mass spectrometrytechniques to resolve and quantify the intact or partially intactglycans. The use of such methods are challenging due to the complexityand number of possible glycan structures present in biological samples.To address the complexity, methods have been developed which employglycan digesting enzymes to liberate and quantify homogenousoligosaccharides generated from the polymeric glycosaminoglycans. Theuse of individual oligosaccharides as the biomarker of disease may notbe sufficient. As a result, an opportunity exists to combineoligosaccharide biomarkers to provide the necessary insight related topresence of disease, prediction of severity, and characterization of theresponse to treatment.

Provided herein is a method of detecting abnormal glycan accumulation,e.g., in human disease. In some instances, the process described hereinincludes a strategy to quantify the changes by measuring the abundanceof all glycans with a disease related glycan residual compound on thenon-reducing end of glycans from a biological sample (e.g.,monosaccharides and/or their modifications such as sulfation,acetylation, phosphorylation, or the like).

Provided in certain embodiments herein are methods of detecting glycanaccumulation in a biological sample, the method comprising:

-   -   a. transforming a glycan of a biological sample with a normally        functioning glycan degradation enzyme to liberate a glycan        residual compound from the non-reducing end of the glycan;    -   b. measuring the amount of the glycan residual compound        liberated by the functioning glycan degradation enzyme with an        analytical device.

In certain embodiments, the method is associated with diagnosing anindividual with abnormal glycan accumulation, or a disorder associatedtherewith.

Therefore, in specific embodiments, provided herein is a method ofdiagnosing an individual as having an abnormal glycan accumulation or adisorder associated with an abnormal glycan accumulation, the methodcomprising:

-   -   a. transforming a glycan of a biological sample with a normally        functioning glycan degradation enzyme to liberate a glycan        residual compound from the non-reducing end of the glycan;    -   b. measuring the amount of the glycan residual compound        liberated by the functioning glycan degradation enzyme with an        analytical device; and    -   c. determining whether the amount of liberated glycan residue is        abnormal.

In certain instances, methods of detecting abnormal glycan accumulationworks based on the observation that altered glycans generated in adisease state are caused by an alteration in the activity of abiosynthetic enzyme (e.g., via increased expression, increased activity,increased substrate, or the like) that leads to the production ofthousands of unique structures.

For example, in certain instances, the induction of an alpha 2,3sialyltransferase leads to the novel expression of thousands ofdifferent glycans (potentially from multiple glycan classes) thatpresent a non-reducing terminal alpha 2,3 linked sialic acid. Byquantifying a limited set of these novel structures using currentmethods, only a fraction of the disease related structures are measured.Instead, as provided in certain embodiments herein, if a samplecontaining glycans (crude or purified for a specific glycan class) istreated with an alpha 2,3 sialidase to liberate the non-reducing endsialic acid, the free sialic acid (non-reducing end glycan residual) canbe measured. This signal would represent a larger portion of thethousands of altered glycan structures that are made in the diseasestate due to the altered expression of the alpha 2,3 sialyltransferase.Furthermore, in certain embodiments, depending on the signal (i.e.,measurement) of the sialic acid liberated, a determination is made as towhether or not the accumulation of sialic acid is abnormal and/orwhether or not such levels of accumulated sialic acid is associated witha disorder.

Another example of the process includes a method involving a biologicalsample containing glycans (purified or not) that is treated with anexo-glycosidase (for example a β-galactosidase). In some of suchembodiments, enzymatic treatment cleaves non-reducing endmonosaccharides within the chosen enzymes specificity (e.g., β-linkedgalactose residues) and liberates them as free monosaccharide (e.g.,galactose). In various embodiments, the free monosaccharide is isolatedand quantified by any analytical method (HPLC, MS, GC, etc), and anydisease that presents changes in the levels of non-reducing end β-linkedgalactose residues is detected or diagnosed.

Similar methods are also optionally utilized in methods of monitoringand/or determining the therapeutic of a treatment or treatment regimen,particularly in the treatment of a disorder associated with abnormalglycan accumulation. For example, provided in certain embodiments hereinis a method of monitoring the treatment of disorders associated with theabnormal degradation, biosynthesis and/or accumulation of glycans, themethods comprising:

-   -   a. following administration of an agent for treating a disorder        associated with the abnormal degradation, biosynthesis and/or        accumulation of glycans to an individual in need thereof, using        an analytical instrument to measure the amount of a population        of a non-reducing end glycan residue present in a transformed        biological sample that has been prepared by:        -   treating a population of glycans, in or isolated from a            biological sample taken from the individual, with at least            one normally functioning glycan degradation enzyme to            liberate non-reducing end glycan residue;    -   b. determining whether or not the amount of liberated        non-reducing end glycan residue has decreased or increased at a        slower rate compared to the amount or rate of increase prior to        administration of the agent for treating a disorder associated        with the abnormal degradation, biosynthesis and/or accumulation        of glycans.

In some embodiments, any process described herein comprises:

-   -   a. comparing an amount of a population of one or more glycan        residual compound present in a transformed biological sample to        an amount of a population of one or more glycan residual        compound present in a control biological sample that has been        treated in a manner substantially similar to the transformed        biological sample.

In certain embodiments, a control biological sample utilized in anyprocess described herein was provided from an individual that does notsuffer from a disorder being diagnosed. In other embodiments, a controlbiological sample is taken from an individual suffering from a disorderbeing diagnosed. In certain embodiments, the result obtained from thecontrol biological sample is stored in a database. In such cases a testsample is optionally compared to a plurality of control data in adatabase. Moreover in certain embodiments, any diagnostic processdescribed herein is optionally utilized alone or in combination withother diagnostic techniques. Other diagnostic techniques include, by wayof non-limiting example, symptom analysis, biopsies, detection ofaccumulation of other compounds in biological samples, or the like. Insome embodiments, control biological samples are optionally taken fromthe same individual at substantially the same time, simply from adifferent location (e.g., one inflamed/arthritic synovial joint fluid vsthe contralateral non-arthritic synovial joint). In other embodiments,control biological samples are optionally taken from the same individualat different points in time (e.g., before therapy and after therapy ifthe method being utilized is a method of monitoring a treatmenttherapy).

In some embodiments, provided herein is a method of monitoring thetreatment of a disorder associated with the abnormal degradation,biosynthesis and/or accumulation of glycans, the method comprising:

-   -   a. following administration of an agent for treating a disorder        associated with the abnormal degradation, biosynthesis and/or        accumulation of glycans to an individual in need thereof, using        an analytical instrument to measure the amount of a population        of a biomarker comprising a non-reducing end glycan residual        compounds present in a transformed biological sample, the        biomarker being generated by treating a population of glycans,        in or isolated from a biological sample from the individual,        with at least one digesting glycan enzyme(s), wherein prior to        enzyme treatment, the biomarker is not present in abundance in        samples from individuals with the disease or condition relative        to individuals without the disease or condition, and    -   b. determining whether or not the amount of biomarker has        decreased or increased at a slower rate compared to the amount        or rate of increase prior to administration of the agent for        treating a disorder associated with the abnormal degradation,        biosynthesis and/or accumulation of glycans.

In some embodiments, the disorder associated with the abnormaldegradation, biosynthesis and/or accumulation of glycans is a lysosomalstorage disease, a cancerous disease, an inflammatory disease, aninfectious disease, a central nervous system disease, or acardiovascular disease.

In some embodiments, the normally functioning glycan degradation enzymeis a glycosidase, sulfatase, phosphorylase, deacetylase, or acombination thereof. In some embodiments, the normally functioningglycan degradation enzyme is a glycosidaseis selected from anexo-glycosidase and an endo-glycosidase. In some embodiments, the glycanresidual compound is a monosaccharide, sulfate, phosphate, acetate, or acombination thereof. In some embodiments, transforming a glycan of abiological sample with a normally functioning glycan degradation enzymecomprises transforming a glycan of a biological sample with a pluralityof normally functioning glycan degradation enzymes. In some embodiments,the glycan is treated with a plurality of normally functioning glycandegradation enzymes concurrently, sequentially, or a combinationthereof.

In some embodiments, prior to measuring the amount of a population ofnon-reducing end glycan residual compounds, the non-reducing end glycanresidual compounds are labeled with a detectable label. In someembodiments, the detectable label is a mass label, a radioisotope label,a fluorescent label, a chromophore label, or affinity label. In someembodiments, the amount of liberated glycan is measured using UV-Visspectroscopy, IR spectroscopy, mass spectrometry, or a combinationthereof.

Provided in certain embodiments herein are:

-   -   a. Methods using a non-reducing end (NRE) glycan biomarker along        with at least one other glycan biomarkers (e.g., a non-reducing        end glycan biomarker, a reducing end biomarker, and/or an        internal glycan biomarker). In some instances, the use of the        two biomarkers provides a very valuable tool that provides        detailed information about disease severity and/or the response        to therapy. In various aspects, the biomarkers detected and/or        analyzed according to the processed described herein are        compared in any suitable manner, e.g., in a ratio or        simultaneous comparison.    -   b. Methods using at least two different glycan biomarkers (e.g.,        wherein each biomarker is individually selected from a        non-reducing end glycan biomarker, a reducing end biomarker, and        an internal glycan biomarker) to identifying or diagnosing        diseases caused by deficiencies in the accumulation and/or        biosynthesis of glycosaminoglycans.        In certain aspects, such methods comprise comparing the amounts        of such biomarkers to each other. In specific embodiments, the        comparison involves determining ratios of the biomarkers,        wherein the biomarkers are glycan fragments generated by        glycosaminoglycan lyase digestion.

Provided in certain embodiments herein is a method of diagnosing anindividual as having a disease or condition associated with abnormalglycan biosynthesis, degradation, or accumulation, the methodcomprising:

-   -   a. generating a biomarker comprising of one or more non-reducing        end glycan residual compound, wherein the biomarker is generated        by treating a population of glycans, in or isolated from a        biological sample from the individual, with at least one        digesting glycan enzymes, wherein prior to enzyme treatment, the        biomarker is not present in abundance in samples from        individuals with the disease or condition relative to        individuals without the disease or condition, and    -   b. using an analytical instrument to detect the presence of        and/or measure the amount of the biomarker produced and        displaying or recording the presence of or a measure of a        population of the biomarker;        wherein the presence of and/or measure the amount of the        biomarker is utilized to determine the presence, identity,        and/or severity of the disease or condition.

In some embodiments, the disease or disorder is caused by an abnormallyfunctioning glycan degradation enzyme and wherein the abnormallyfunctioning glycan degradation enzyme and the normally functioningglycan degradation enzyme are of the same type. In some embodiments, theabnormally functioning glycan degradation enzyme functions abnormally asa result of being present in an abnormally low amount, functioningimproperly, or a combination thereof. In some embodiments, the abnormalglycan accumulation comprises the accumulation of abnormal amounts ofglycans. In some embodiments, the abnormal glycan accumulation comprisesthe accumulation of abnormal amounts of normal glycans. In someembodiments, the abnormal glycan accumulation comprises the accumulationof abnormal amounts of abnormal glycans.

In some embodiments, the normally functioning glycan degradation enzymeis a glycosidase, sulfatase, phosphorylase, deacetylase, or acombination thereof. In some embodiments, the normally functioningglycan degradation enzyme is a glycosidase selected from anexo-glycosidase and an endo-glycosidase. In some embodiments, theglycosidase is an exo-glycosidase selected from the group consisting ofa galactosidase, and a glucuronidase. In some embodiments, the glycanresidual compound is a monosaccharide. In some embodiments, the glycanresidual compound is sulfate, phosphate, acetate, or a combinationthereof.

In some embodiments, a biological sample is purified prior totransforming a glycan thereof. In some embodiments, the process ofpurifying a biological sample comprises removing monosaccharidestherefrom, removing sulfates therefrom, removing phosphates therefrom,removing acetate therefrom, or a combination thereof. In someembodiments, transforming a glycan of a biological sample with anormally functioning glycan degradation enzyme comprises transforming aglycan of a biological sample with a plurality of normally functioningglycan degradation enzymes. In some embodiments, the glycan is treatedwith a plurality of normally functioning glycan degradation enzymesconcurrently, sequentially, or a combination thereof.

In some embodiments, the disorder associated with an abnormal glycanaccumulation is MPS I, MPS II, MPS IIIA, MPS IVA, MPSVI, or FabryDisease.

In some embodiments, determining whether the amount of liberated glycanresidue is abnormal comprises labeling the glycan residue with adetectable label and measuring the amount of labeled glycan residue withan analytical instrument. In some embodiments, the detectable label is amass label, a radioisotope label, a fluorescent label, a chromophorelabel, or affinity label. In some embodiments, the amount of liberatedglycan is measured using UV-Vis spectroscopy, IR spectroscopy, massspectrometry, or a combination thereof.

Provided herein, in certain embodiments, is a method of diagnosing anindividual as having a disease or condition (e.g., associated withabnormal glycan biosynthesis, degradation, or accumulation), the methodcomprising:

-   -   a. generating a first biomarker comprising a glycan residual        compound, wherein the first biomarker is generated by treating a        population of glycans, in or isolated from a biological sample        from the individual, with at least one digesting glycan enzyme,        wherein prior to enzyme treatment, the first biomarker is not        present in abundance in samples from individuals with the        disease or condition relative to individuals without the disease        or condition,    -   b. generating a second biomarker comprising a glycan residual        compound, wherein the second biomarker is generated by treating        a population of glycans, in or isolated from a biological sample        from the individual, with at least one digesting glycan enzyme        in the same or different digestion step as provided in step (a),        wherein prior to enzyme treatment, the second biomarker is not        present in abundance in samples from individuals with the        disease or condition relative to individuals without the disease        or condition,    -   c. using an analytical instrument to detect the presence of        and/or measure the amount of the first and second biomarker        produced and displaying or recording the presence of or a        measure of a population of the first and second biomarkers, and    -   d. monitoring and/or comparing the amounts of the first and        second biomarkers in a biological sample;        wherein the presence of and/or measure of the amounts of the        first and second biomarkers are utilized to determine the        presence, identity, and/or severity of the disease or condition.

In some embodiments, the first biomarker is a non-reducing end glycanresidual compound. In some embodiments, the disease or disorder iscaused by an abnormally functioning glycan degradation enzyme andwherein the abnormally functioning glycan degradation enzyme and thedigesting glycan enzyme are of the same type. In some embodiments, thenon-reducing end glycan residual compound is a monosaccharide. In someembodiments, the non-reducing end glycan residual compound is not amonosaccharide.

In some embodiments, the second biomarker is derived or generated fromthe reducing end of the same glycan from which the first non-reducingend glycan residual compound biomarker was generated. In someembodiments, the second biomarker is derived or generated from theinternal oligosaccharide structures of the same glycan from which thefirst non-reducing end glycan residual compound biomarker was generated.In some embodiments, the disease or disorder is caused by the abnormalfunction of a glycan degradation enzyme in the individual, and whereinthe second biomarker can be generated by treating the first non-reducingend glycan residual compound biomarker with the glycan degradationenzyme that is functioning abnormally in the individual.

In some embodiments, the disease or condition associated with abnormalglycan biosynthesis, degradation, or accumulation is a lysosomal storagedisease. In some embodiments, the lysosomal storage disease isMucopolysaccharidosis. In some embodiments, the Mucopolysaccharidosis isMPS I, II, IIIA, IIIB, IIIC, IIID, IVA, IVB, VI, or VII. In someembodiments, the disease or condition associated with abnormal glycanbiosynthesis, degradation, or accumulation is MetachromaticLeukodystrophy or Krabbe disease. In some embodiments, the disease orcondition associated with abnormal glycan biosynthesis, degradation, oraccumulation is Gangliosidosis. In some embodiments, the Gangliosidosisis Tay Sachs, Sandhoff, AB Variant, or GM-1 Gangliosidoses.

In some embodiments, the presence of and/or measure of the firstnon-reducing end glycan residual compound biomarker in combination withor in relation to the second biomarker is utilized to monitor thetreatment of a disorder associated with the abnormal biosynthesis ofglycans. In some embodiments, the presence of and/or measure of thefirst non-reducing end glycan residual compound biomarker in combinationwith or in relation to the second biomarker is utilized to monitor thetreatment of a disorder associated with the abnormal degradation oraccumulation of glycans. In some embodiments, the treatment is enzymereplacement therapy. In some embodiments, the absence of an increase inthe second biomarker combined with a reduction in the non-reducing endglycan residual compound biomarker indicates a positive response totreatment of the disorder associated with abnormal degradation oraccumulation of glycans.

Glycan Accumulation:

In various instances, glycan accumulation occurs in a biological sampleas a result natural glycan biosynthetic and/or degradation processes. Insome instances, abnormal glycan accumulation occurs in a biologicalsample as a result of a disorder or disease within an individual fromwhich the biological sample is obtained.

In certain embodiments, abnormal glycan accumulation that is observableby methods described herein is associated with the accumulation ofglycans in a manner that does not normally occur in individuals who arenot in a disease state.

In some embodiments, such accumulation includes the accumulation ofabnormal glycans. In certain instances, these abnormal glycans includeglycans that are not normally produced in an individual, or a particularbiological sample thereof, in the absence of a particular disease state.Therefore, in some embodiments, abnormal glycan accumulation includesthe accumulation of glycans, the glycans being abnormal themselves,especially in any significant quantity. In other words, such glycans areabnormal glycans in individuals or particular biological samples thereofwhen such individuals are in a non-diseased, normal, or wild type state.

In some embodiments, such accumulation includes the abnormalaccumulation of glycans. In some instances, these glycans are glycansthat normally occur in individuals in a non-diseased state, but at loweror higher levels or are abnormal only due to the location wherein theyare produced. Therefore, in some embodiments, abnormal glycanaccumulation includes the accumulation of abnormal amounts of glycans orthe location thereof, the glycans being normally occurring or abnormalglycans. In other words, the amount of glycan accumulation is abnormalin individuals, or particular biological samples thereof, when suchindividuals are in a non-diseased, normal, or wild type state.

Biological Sample:

Biological samples suitable for analysis according to the methods andprocesses described herein include, by way of non-limiting example,blood, serum, urine, hair, saliva, skin, tissue, plasma, cerebrospinalfluid (CSF), amniotic fluid, nipple aspirate, sputum, tears, lungaspirate, semen, feces, synovial fluid, nails, or the like. In specificembodiments, the biological samples suitable for analysis according tothe methods and processes described herein include, by way ofnon-limiting example, urine, serum, plasma, or CSF. In certainembodiments, processes for detecting glycan in a sample compriseproviding, from the individual, a test biological sample that comprisesglycan. In some embodiments, providing a test biological sample from anindividual includes obtaining the sample from the individual orobtaining the sample from another source (e.g., from a technician orinstitution that obtained the sample from the individual). In someembodiments, the biological sample is obtained from any suitable source,e.g., any tissue or cell (e.g., urine, serum, plasma, or CSF) of anindividual. In certain embodiments, the tissue and/or cell from whichthe glycans are recovered is obtained from liver tissue or cells, braintissue or cells, kidney tissue or cells, or the like.

In certain embodiments, a biological sample according to any processdescribed herein is taken from any individual. In some embodiments, theindividual is an individual suspected of suffering from a disorderassociated with abnormal glycan accumulation, biosynthesis, and/ordegradation. In certain embodiments, the individual is a newborn orfetus.

In some embodiments, provided herein is a composition comprisingisolated glycans, wherein the glycans were isolated from a biologicalsample, and one or more glycan degradation enzyme. In certainembodiments, the composition further comprises one or more biomarkergenerated according to any method described herein (e.g., wherein thebiomarker is a non-reducing end glycan residual compound). In certainembodiments, provided herein is a biomarker described herein (e.g., alabeled or non-labeled non-reducing end glycan residual compound) and ananalytical instrument or chromatographic resin.

Degradation Enzymes:

In certain embodiments, any suitable enzyme is optionally utilized inorder to remove a glycan residual compound from the non-reducing end ofa glycan. In certain disorders, e.g., as described herein, various typesof abnormal glycan accumulation occurs. In certain instances, this typeof glycan accumulation is detected and/or measured utilizing anysuitable enzyme, e.g., as described herein. For example, Tables 1-4illustrate various enzymes that are utilized in various embodiments ofthe processes described herein. Any enzyme with the desired specificityis optionally utilized in any process herein (i.e., to liberate thenon-reducing end structures). Enzymes suitable for use in the processesdescribed herein include, by way of non-limiting example, eukaryotic,prokaryotic, native, or recombinant enzymes.

In certain embodiments, a disorder associated with abnormal glycanaccumulation includes a disorder associated therewith is caused by anabnormally functioning glycan degradation enzyme. In variousembodiments, the abnormally functioning glycan degradation enzymefunctions abnormally as a result of being present in an abnormally lowamount, functioning improperly, or a combination thereof. For example,an abnormally functioning glycan degradation enzyme functions abnormallyas a result of being present in an amount of less than 50%, less than40%, less than 30%, less than 20%, less than 10%, or less than 5% thanis present in an individual with normal amounts of the glycandegradation enzyme (e.g., an individual in a non-diseased, normal, orwild type state). In further or alternative embodiments, abnormallyfunctioning glycan degradation enzymes are present in a normal amount,but do not function properly in degrading glycans. For example, suchenzymes may be have amino acid substitutions in the sequences thereofthat reduce or eliminate the glycan degradative properties of theenzyme.

In some embodiments, wherein abnormal glycan accumulation results, atleast partially from, an abnormally functioning glycan degradationenzyme, a normally functioning glycan degradation is optionallyutilized, particularly wherein the abnormally functioning glycandegradation enzyme and the normally functioning glycan degradationenzyme are of the same type.

Normally functioning glycan degradation enzymes that are used in variousembodiments described herein include, by way of non-limiting example,glycosidases, sulfatases, phosphorylases, deacetylases, sialidases, orcombinations thereof. In more specific embodiments, a normallyfunctioning glycan degradation enzyme is a glycosidase, e.g., anexo-glycosidase or an endo-glycosidase. In more specific embodiments,the glycosidase is an exo-glycosidase, e.g., galactosidase, and aglucuronidase. In some embodiments, such enzymes serve to remove variousglycan residual compounds, such as, monosaccharides, sulfate, phosphate,acetate, sialic acid, or combinations thereof, which are detected and/ormeasured in methods described herein.

In certain embodiments, one or normally functioning glycan degradationenzyme is optionally utilized to liberate a targeted glycan residualcompound. Multiple enzyme treatments of glycans within a biologicalsample are useful in various embodiments, e.g., wherein a particularenzyme is unable to liberate a targeted residual glycan compound withoutfirst modifying the non-reducing end of the glycan. For example, a firstenzyme is optionally utilized to remove a sulfate so that a secondenzyme can be utilized to remove a monosaccharide. In variousembodiments, the glycans are treated with a plurality of normallyfunctioning glycan degradation enzymes concurrently, sequentially, or acombination thereof.

Various enzymes that are used in various embodiments of the methodsdescribed herein include, by way of non-limiting example, a glycosidase.Non-limiting examples of glycosidase that are optionally utilized in themethods described herein include, by way of non-limiting example,enzymes categorized as 3.2.1.X by BRENDA (the comprehensive EnzymeInformation System) including 3.2.1.1 alpha-amylase, 3.2.1.B1extracellular agarase, 3.2.1.2 beta-amylase, 3.2.1.3 glucan1,4-alpha-glucosidase, 3.2.1.4 cellulase, 3.2.1.5 licheninase, 3.2.1.6endo-1,3(4)-beta-glucanase, 3.2.1.7 inulinase, 3.2.1.8endo-1,4-beta-xylanase, 3.2.1.9 amylopectin-1,6-glucosidase, 3.2.1.10oligo-1,6-glucosidase, 3.2.1.11 dextranase, 3.2.1.12cycloheptaglucanase, 3.2.1.13 cyclohexaglucanase, 3.2.1.14 chitinase,3.2.1.15 polygalacturonase, 3.2.1.16 alginase, 3.2.1.17 lysozyme,3.2.1.18 exo-alpha-sialidase, 3.2.1.19 heparinase, 3.2.1.20alpha-glucosidase, 3.2.1.21 beta-glucosidase, 3.2.1.22alpha-galactosidase, 3.2.1.23 beta-galactosidase, 3.2.1.24alpha-mannosidase, 3.2.1.25 beta-mannosidase, 3.2.1.26beta-fructofuranosidase, 3.2.1.27 alpha-1,3-glucosidase, 3.2.1.28alpha,alpha-trehalase, 3.2.1.29 chitobiase, 3.2.1.30beta-D-acetylglucosaminidase, 3.2.1.31 beta-glucuronidase, 3.2.1.32xylan endo-1,3-beta-xylosidase, 3.2.1.33 amylo-alpha-1,6-glucosidase,3.2.1.34 chondroitinase, 3.2.1.35 hyaluronoglucosaminidase, 3.2.1.36hyaluronoglucuronidase, 3.2.1.37 xylan 1,4-beta-xylosidase, 3.2.1.38beta-D-fucosidase, 3.2.1.39 glucan endo-1,3-beta-D-glucosidase, 3.2.1.40alpha-L-rhamnosidase, 3.2.1.41 pullulanase, 3.2.1.42 GDP-glucosidase,3.2.1.43 beta-L-rhamnosidase, 3.2.1.44 fucoidanase, 3.2.1.45glucosylceramidase, 3.2.1.46 galactosylceramidase, 3.2.1.47galactosylgalactosylglucosylceramidase, 3.2.1.48 sucrosealpha-glucosidase, 3.2.1.49 alpha-N-acetylgalactosaminidase, 3.2.1.50alpha-N-acetylglucosaminidase, 3.2.1.51 alpha-L-fucosidase, 3.2.1.52beta-N-acetylhexosaminidase, 3.2.1.53 beta-N-acetylgalactosaminidase,3.2.1.54 cyclomaltodextrinase, 3.2.1.55 alpha-N-arabinofuranosidase,3.2.1.56 glucuronosyl-disulfoglucosamine glucuronidase, 3.2.1.57isopullulanase, 3.2.1.58 glucan 1,3-beta-glucosidase, 3.2.1.59 glucanendo-1,3-alpha-glucosidase, 3.2.1.60 glucan1,4-alpha-maltotetraohydrolase, 3.2.1.61 mycodextranase, 3.2.1.62glycosylceramidase, 3.2.1.63 1,2-alpha-L-fucosidase, 3.2.1.642,6-beta-fructan 6-levanbiohydrolase, 3.2.1.65 levanase, 3.2.1.66quercitrinase, 3.2.1.67 galacturan 1,4-alpha-galacturonidase, 3.2.1.68isoamylase, 3.2.1.69 amylopectin 6-glucanohydrolase, 3.2.1.70 glucan1,6-alpha-glucosidase, 3.2.1.71 glucan endo-1,2-beta-glucosidase,3.2.1.72 xylan 1,3-beta-xylosidase, 3.2.1.73 licheninase, 3.2.1.74glucan 1,4-beta-glucosidase, 3.2.1.75 glucan endo-1,6-beta-glucosidase,3.2.1.76 L-iduronidase, 3.2.1.77 mannan 1,2-(1,3)-alpha-mannosidase,3.2.1.78 mannan endo-1,4-beta-mannosidase, 3.2.1.79alpha-L-arabinofuranoside hydrolase, 3.2.1.80 fructan beta-fructosidase,3.2.1.81 beta-agarase, 3.2.1.82 exo-poly-alpha-galacturonosidase,3.2.1.83 kappa-carrageenase, 3.2.1.84 glucan 1,3-alpha-glucosidase,3.2.1.85 6-phospho-beta-galactosidase, 3.2.1.866-phospho-beta-glucosidase, 3.2.1.87 capsular-polysaccharideendo-1,3-alpha-galactosidase, 3.2.1.88 beta-L-arabinosidase, 3.2.1.89arabinogalactan endo-1,4-beta-galactosidase, 3.2.1.90 arabinogalactanendo-1,3-beta-galactosidase, 3.2.1.91 cellulose 1,4-beta-cellobiosidase,3.2.1.92 peptidoglycan beta-N-acetylmuramidase, 3.2.1.93alpha,alpha-phosphotrehalase, 3.2.1.94 glucan 1,6-alpha-isomaltosidase,3.2.1.95 dextran 1,6-alpha-isomaltotriosidase, 3.2.1.96mannosylglycoprotein endo-beta-N-acetylglucosaminidase, 3.2.1.97glycopeptide alpha-N-acetylgalactosaminidase, 3.2.1.98 glucan1,4-alpha-maltohexaosidase, 3.2.1.99 arabinanendo-1,5-alpha-L-arabinosidase, 3.2.1.100 mannan 1,4-mannobiosidase,3.2.1.101 mannan endo-1,6-alpha-mannosidase, 3.2.1.102blood-group-substance endo-1,4-beta-galactosidase, 3.2.1.103keratan-sulfate endo-1,4-beta-galactosidase, 3.2.1.104steryl-beta-glucosidase, 3.2.1.105 3alpha(S)-strictosidinebeta-glucosidase, 3.2.1.106 mannosyl-oligosaccharide glucosidase,3.2.1.107 protein-glucosylgalactosylhydroxylysine glucosidase, 3.2.1.108lactase, 3.2.1.109 endogalactosaminidase, 3.2.1.110 mucinaminylserinemucinaminidase, 3.2.1.111 1,3-alpha-L-fucosidase, 3.2.1.1122-deoxyglucosidase, 3.2.1.113 mannosyl-oligosaccharide1,2-alpha-mannosidase, 3.2.1.114 mannosyl-oligosaccharide1,3-1,6-alpha-mannosidase, 3.2.1.115 branched-dextranexo-1,2-alpha-glucosidase, 3.2.1.116 glucan1,4-alpha-maltotriohydrolase, 3.2.1.117 amygdalin beta-glucosidase,3.2.1.118 prunasin beta-glucosidase, 3.2.1.119 vicianinbeta-glucosidase, 3.2.1.120 oligoxyloglucan beta-glycosidase, 3.2.1.121polymannuronate hydrolase, 3.2.1.122 maltose-6′-phosphate glucosidase,3.2.1.123 endoglycosylceramidase, 3.2.1.124 3-deoxy-2-octulosonidase,3.2.1.125 raucaffricine beta-glucosidase, 3.2.1.126 coniferinbeta-glucosidase, 3.2.1.127 1,6-alpha-L-fucosidase, 3.2.1.128glycyrrhizinate beta-glucuronidase, 3.2.1.129 endo-alpha-sialidase,3.2.1.130 glycoprotein endo-alpha-1,2-mannosidase, 3.2.1.131 xylanalpha-1,2-glucuronosidase, 3.2.1.132 chitosanase, 3.2.1.133 glucan1,4-alpha-maltohydrolase, 3.2.1.134 difructose-anhydride synthase,3.2.1.135 neopullulanase, 3.2.1.136 glucuronoarabinoxylanendo-1,4-beta-xylanase, 3.2.1.137 mannan exo-1,2-1,6-alpha-mannosidase,3.2.1.138 anhydrosialidase, 3.2.1.139 alpha-glucuronidase, 3.2.1.140lacto-N-biosidase, 3.2.1.141 4-alpha-D-{(1->4)-alpha-D-glucano}trehalosetrehalohydrolase, 3.2.1.142 limit dextrinase, 3.2.1.143 poly(ADP-ribose)glycohydrolase, 3.2.1.144 3-deoxyoctulosonase, 3.2.1.145 galactan1,3-beta-galactosidase, 3.2.1.146 beta-galactofuranosidase, 3.2.1.147thioglucosidase, 3.2.1.148 ribosylhomocysteinase, 3.2.1.149beta-primeverosidase, 3.2.1.150 oligoxyloglucan reducing-end-specificcellobiohydrolase, 3.2.1.151 xyloglucan-specificendo-beta-1,4-glucanase, 3.2.1.152 mannosylglycoproteinendo-beta-mannosidase, 3.2.1.153 fructan beta-(2,1)-fructosidase,3.2.1.154 fructan beta-(2,6)-fructosidase, 3.2.1.155 xyloglucan-specificexo-beta-1,4-glucanase, 3.2.1.156 oligosaccharide reducing-end xylanase,3.2.1.157 iota-carrageenase 3.2.1.158 alpha-agarase, 3.2.1.159alpha-neoagaro-oligosaccharide hydrolase, 3.2.1.160 xyloglucan-specificexo-beta-1,4-glucanase, 3.2.1.161 beta-apiosyl-beta-glucosidase,3.2.1.162 lambda-carrageenase, 3.2.1.163 1,6-alpha-D-mannosidase,3.2.1.164 galactan endo-1,6-beta-galactosidase, 3.2.1.165exo-1,4-beta-D-glucosaminidase, or a combination thereof.

Other enzymes that are used in various embodiments of the methodsdescribed herein include, by way of non-limiting example, a sulfataseincluding, e.g., enzymes categorized as 3.1.6.X by BRENDA (thecomprehensive Enzyme Information System) including 3.1.6.1arylsulfatase, 3.1.6.2 steryl-sulfatase, 3.1.6.3 glycosulfatase, 3.1.6.4N-acetylgalactosamine-6-sulfatase, 3.1.6.5 sinigrin sulfohydrolase;myrosulfatase, 3.1.6.6 choline-sulfatase, 3.1.6.7cellulose-polysulfatase, 3.1.6.8 cerebroside-sulfatase, 3.1.6.9chondro-4-sulfatase, 3.1.6.10 chondro-6-sulfatase, 3.1.6.11 disulfoglucosamine-6-sulfatase, 3.1.6.12N-acetylgalactosamine-4-sulfatase, 3.1.6.13 iduronate-2-sulfatase,3.1.6.14 N-acetylglucosamine-6-sulfatase, 3.1.6.15N-sulfoglucosamine-3-sulfatase, 3.1.6.16 monomethyl-sulfatase, 3.1.6.17D-lactate-2-sulfatase, 3.1.6.18 glucuronate-2-sulfatase, 3.10.1.1N-sulfoglucosamine sulfohydrolase, or combinations thereof.

Certain enzymes that are used in various embodiments of the methodsdescribed herein include, by way of non-limiting example, a deacetylase,e.g., an exo-deacetylase, including, by way of non-limiting example, thealpha-glucosaminide N-acetyltransferase (2.3.1.78) or similar enzymes.

Certain enzymes that are used in various embodiments of the methodsdescribed herein include, by way of non-limiting example, a carbohydratephosphatase including, e.g., 3.1.3.1 alkaline phosphatase, 3.1.3.2 acidphosphatase, 3.1.3.B2 diacylglycerol pyrophosphate phosphatase, 3.1.3.3phosphoserine phosphatase, 3.1.3.4 phosphatidate phosphatase, 3.1.3.55′-nucleotidase, 3.1.3.6 3′-nucleotidase, 3.1.3.7 3′(2′),5′-bisphosphatenucleotidase, 3.1.3.8 3-phytase, 3.1.3.9 glucose-6-phosphatase, 3.1.3.10glucose-1-phosphatase, 3.1.3.11 fructose-bisphosphatase, 3.1.3.12trehalose-phosphatase, 3.1.3.13 bisphosphoglycerate phosphatase,3.1.3.14 methylphosphothioglycerate phosphatase, 3.1.3.15histidinol-phosphatase, 3.1.3.16 phosphoprotein phosphatase, 3.1.3.17[phosphorylase]phosphatase, 3.1.3.18 phosphoglycolate phosphatase,3.1.3.19 glycerol-2-phosphatase, 3.1.3.20 phosphoglycerate phosphatase,3.1.3.21 glycerol-1-phosphatase, 3.1.3.22 mannitol-1-phosphatase,3.1.3.23 sugar-phosphatase, 3.1.3.24 sucrose-phosphate phosphatase,3.1.3.25 inositol-phosphate phosphatase, 3.1.3.26 4-phytase, 3.1.3.27phosphatidylglycerophosphatase, 3.1.3.28 ADP-phosphoglyceratephosphatase, 3.1.3.29 N-acylneuraminate-9-phosphatase, 3.1.3.303′-phosphoadenylylsulfate 3′-phosphatase, 3.1.3.31 nucleotidase,3.1.3.32 polynucleotide 3′-phosphatase, 3.1.3.33 polynucleotide5′-phosphatase, 3.1.3.34 deoxynucleotide 3′-phosphatase, 3.1.3.35thymidylate 5′-phosphatase, 3.1.3.36 phosphoinositide 5-phosphatase,3.1.3.37 sedoheptulose-bisphosphatase, 3.1.3.38 3-phosphoglyceratephosphatase, 3.1.3.39 streptomycin-6-phosphatase, 3.1.3.40guanidinodeoxy-scyllo-inositol-4-phosphatase, 3.1.3.414-nitrophenylphosphatase, 3.1.3.42 [glycogen-synthase-D]phosphatase,3.1.3.43 [pyruvate dehydrogenase (acetyl-transferring)]-phosphatase,3.1.3.44 [acetyl-CoA carboxylase]-phosphatase, 3.1.3.453-deoxy-manno-octulosonate-8-phosphatase, 3.1.3.46fructose-2,6-bisphosphate 2-phosphatase, 3.1.3.47[hydroxymethylglutaryl-CoA reductase (NADPH)]-phosphatase, 3.1.3.48protein-tyrosine-phosphatase, 3.1.3.49 [pyruvate kinase]-phosphatase,3.1.3.50 sorbitol-6-phosphatase, 3.1.3.51 dolichyl-phosphatase, 3.1.3.52[3-methyl-2-oxobutanoate dehydrogenase(2-methylpropanoyl-transferring)]-phosphatase, 3.1.3.53[myosin-light-chain] phosphatase, 3.1.3.54 fructose-2,6-bisphosphate6-phosphatase, 3.1.3.55 caldesmon-phosphatase, 3.1.3.56inositol-polyphosphate 5-phosphatase, 3.1.3.57 inositol-1,4-bisphosphate1-phosphatase, 3.1.3.58 sugar-terminal-phosphatase, 3.1.3.59alkylacetylglycerophosphatase, 3.1.3.60 phosphoenolpyruvate phosphatase,3.1.3.61 inositol-1,4,5-trisphosphate 1-phosphatase, 3.1.3.62 multipleinositol-polyphosphate phosphatase, 3.1.3.632-carboxy-D-arabinitol-1-phosphatase, 3.1.3.64phosphatidylinositol-3-phosphatase, 3.1.3.65 inositol-1,3-bisphosphate3-phosphatase, 3.1.3.66 phosphatidylinositol-3,4-bisphosphate4-phosphatase, 3.1.3.67 phosphatidylinositol-3,4,5-trisphosphate3-phosphatase, 3.1.3.68 2-deoxyglucose-6-phosphatase, 3.1.3.69glucosylglycerol 3-phosphatase, 3.1.3.70 mannosyl-3-phosphoglyceratephosphatase, 3.1.3.71 2-phosphosulfolactate phosphatase, 3.1.3.725-phytase, 3.1.3.73 alpha-ribazole phosphatase, 3.1.3.74 pyridoxalphosphatase, 3.1.3.75 phosphoethanolamine/phosphocholine phosphatase,3.1.3.76 lipid-phosphate phosphatase, 3.1.3.77 acireductone synthase,3.1.3.78 phosphatidylinositol-4,5-bisphosphate 4-phosphatase, or3.1.3.79 mannosylfructose-phosphate phosphatase, or a combinationthereof.

In some embodiments, processes described herein include incubation anddigestion with a first enzyme to clear a specific non-reducing endstructure, incubation and digestion with a second enzyme. In certainembodiments, this multi-enzyme approach is useful in order to reduce thebackground. For example, in MPS II treating the sample with aniduronidase and/or glucuronidase to clear all non-sulfated non-reducingend uronic acids (this enzyme will not cleave sulfated iduronic acids)before 2-O sulfatase treatment. This approach will clear allnon-sulfated non-reducing end uronic acids so that upon desulfation withthe 2-O sulfatase the newly releasable uronic acids will be those thatwere previously sulfated (and therefore resistant to the action of theiduronidase and/or glucuronidase).

Glycan Residual Compounds:

Glycan residual compounds detected, measured, analyzed, and/or otherwisecharacterized according to any process described herein include anysuitable glycan residue that is liberated from the non-reducing end of aglycan (e.g., a glycan obtained from a biological sample of anindividual). In specific instances, glycan residual compounds including,e.g., oligosaccharides, monosaccharides, sulfate, phosphate, sialicacid, acetate, or the like. Specific glycan residual compounds useful inany process herein are described in Tables 1-4.

In some embodiments, the generated biomarker is a glycan residualcompound. In some embodiments, the glycan residual compound is amonosaccharide. In certain embodiments, the glycan residual compound issulfate, phosphate, acetate, or a combination thereof. In certainembodiments, the glycan residual compound has a molecular weight of lessthan 2000 g/mol, less than 1500 g/mol, less than 1000 g/mol, less than500 g/mol, less than 400 g/mol, less than 300 g/mol, less than 260g/mol, less than 200 g/mol, less than 100 g/mol, or the like (e.g.,prior to tagging with any detectable label that may be included in aprocess described herein).

Biomarker Ratios:

In various aspects provided herein, the simultaneous measurement orratios of various biomarkers (e.g., saturated non-reducing endstructures or internal unsaturated disaccharides generated by enzymaticdepolymerization of glycosaminoglycans) reveals information aboutdisease severity and response to therapy. Depending on the specificdisease being diagnosed or otherwise analyzed, these comparisons (e.g.,simultaneous measurement or ratios) use varying saturated andunsaturated structures.

For example, in some embodiments, one or more of the biomarkers used inany process described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 1. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS I. In someembodiments, the first and second biomarkers are biomarkers of Table 1.In other embodiments, the first biomarker is a biomarker of Table 1 andthe second biomarker is a biomarker of Table 9, 3, 4, or 6. In furtheror alternative embodiments, both the first and second biomarkers arefrom Table 1.

TABLE 1 HS derived CS/DS derived IdoA-GlcNS IdoA-GalNAc4S IdoA-GlcNS6SIdoA-GalNAc6S IdoA-GlcNAc IdoA-GalNAc IdoA-GlcNAc6S IdoA-GalNAc4S6S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 2. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS II. In someembodiments, the first biomarker is a biomarker of Table 2 and thesecond biomarker is a biomarker of Table 1. In other embodiments, thefirst biomarker is a biomarker of Table 2 and the second biomarker is abiomarker of Table 9. In further or alternative embodiments, both thefirst and second biomarkers are from Table 2.

TABLE 2 HS derived CS/DS derived IdoA2S-GlcNS IdoA2S-GalNAc4SIdoA2S-GlcNS6S IdoA2S-GalNAc6S IdoA2S-GlcNAc IdoA2S-GalNAcIdoA2S-GlcNAc6S IdoA2S-GalNAc4S6S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 3. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS IIIA. In someembodiments, the first biomarker is a biomarker of Table 3 and thesecond biomarker is a biomarker of Table 5. In other embodiments, thefirst biomarker is a biomarker of Table 3 and the second biomarker is abiomarker of Table 9. In further or alternative embodiments, both thefirst and second biomarkers are from Table 3.

TABLE 3 HS derived GlcNS GlcNS +/− 6S-UA +/− 2S-GlcNAc +/− 6S GlcNS +/−6S-UA +/− 2S-GlcNS +/− 6S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 4. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS IIIB. In someembodiments, the first biomarker is a biomarker of Table 4 and thesecond biomarker is a biomarker of Table 1, 2, or 8. In otherembodiments, the first biomarker is a biomarker of Table 4 and thesecond biomarker is a biomarker of Table 9. In further or alternativeembodiments, both the first and second biomarkers are from Table 4.

TABLE 4 HS derived GlcNAc GlcNAc-UA +/− 2S-GlcNAc +/− 6S GlcNAc-UA +/−2S-GlcNS +/− 6S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 5. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS IIIC. In someembodiments, the first biomarker is a biomarker of Table 5 and thesecond biomarker is a biomarker of Table 4. In other embodiments, thefirst biomarker is a biomarker of Table 5 and the second biomarker is abiomarker of Table 9. In further or alternative embodiments, both thefirst and second biomarkers are from Table 5.

TABLE 5 HS derived GlcN GlcN +/− 6S-UA +/− 2S-GlcNAc +/− 6S GlcN +/−6S-UA +/− 2S-GlcNS +/− 6S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 6. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS IIID. In someembodiments, the first biomarker is a biomarker of Table 6 and thesecond biomarker is a biomarker of Table 4 or 5. In other embodiments,the first biomarker is a biomarker of Table 6 and the second biomarkeris a biomarker of Table 9. In further or alternative embodiments, boththe first and second biomarkers are from Table 6.

TABLE 6 HS derived GlcN6S GlcNAc6S GlcN6S-UA +/− 2S-GlcNAc +/− 6SGlcNAc6S-UA +/− 2S-GlcNAc +/− 6S GlcN6S-UA +/− 2S-GlcNS +/− 6SGlcNAc6S-UA +/− 2S-GlcNS +/− 6S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 7. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS VI. In someembodiments, the first biomarker is a biomarker of Table 7 and thesecond biomarker is a biomarker of Table 8. In other embodiments, thefirst biomarker is a biomarker of Table 7 and the second biomarker is abiomarker of Table 9. In further or alternative embodiments, both thefirst and second biomarkers are from Table 7.

TABLE 7 CS derived GalNAc4S GalNAc4S-UA-GalNAc GalNAc4S-UA-GalNAc4SGalNAc4S-UA-GalNAc6S GalNAc4S-UA-GalNAc4S6S GalNAc4S-UA2S-GalNAcGalNAc4S-UA2S-GalNAc4S GalNAc4S-UA2S-GalNAc6S GalNAc4S-UA2S-GalNAc4S6S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 8. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS VII. In someembodiments, the first biomarker is a biomarker of Table 8 and thesecond biomarker is a biomarker of Table 3, 4, 6, or 7. In otherembodiments, the first biomarker is a biomarker of Table 8 and thesecond biomarker is a biomarker of Table 9. In further or alternativeembodiments, both the first and second biomarkers are from Table 1.

TABLE 8 HS derived CS derived KS derived GlcA-GlcNAc GlcA-GalNAcGal-GlcNAc GlcA-GlcNS GlcA-GalNAc4S Gal-GlcNAc6S GlcA-GlcNAc6SGlcA-GalNAc6S Gal6S-GlcNAc GlcA-GlcNS6S GlcA-GalNAc4S6S Gal6S-GlcNAc6SGlcNAc-Gal GlcNAc-Gal6S GlcNAc6S-Gal GlcNAc6S-Gal6S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the unsaturated biomarkers(glycan fragments) set forth in Table 9. In further or alternativeembodiments, both the first and second biomarkers are from Table 9.

TABLE 9 HS derived CS derived ΔUA-GlcN ΔUA-GalNAc ΔUA-GlcN6SΔUA-GalNAc4S ΔUA2S-GlcN ΔUA-GalNAc6S ΔUAS-GlcN6S ΔUA2S-GalNAc ΔUA-GlcNAcΔUA2S-GalNAc4S ΔUA-GlcNAc6S ΔUA2S-GalNAc6S ΔUA2S-GlcNAc ΔUA-GalNAc4S6SΔUA2S-GlcNAc6S ΔUA2S-GalNAc4S6S ΔUA-GlcNS ΔUA-GlcNS6S ΔUA-GlcNS3SΔUA2S-GlcNS ΔUA2S-GlcNS6S ΔUA2S-GlcNS3S ΔUA-GlcNS6S3S ΔUA2S-GlcNS6S3S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 10. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS IVA. In someembodiments, the first biomarker is a biomarker of Table 10 and thesecond biomarker is a biomarker of Table 8. In other embodiments, thefirst biomarker is a biomarker of Table 10 and the second biomarker is abiomarker of Table 9. In further or alternative embodiments, both thefirst and second biomarkers are from Table 10.

TABLE 10 KS derived CS Derived Gal6S GalNAc6S Gal6S-GlcNAc GalNAc6S4SGal6S-GlcNAc6S GalNAc6S-UA-GalNAc Gal6S-GlcNAc-Gal GalNAc6S-UA-GalNAc4SGal6S-GlcNAc-Gal6S GalNAc6S-UA-GalNAc6S Gal6S-GlcNAc6S-GalGalNAc6S-UA-GalNAc4S6S Gal6S-GlcNAc6S-Gal6S GalNAc6S-UA2S-GalNAcGalNAc6S-UA2S-GalNAc4S GalNAc6S-UA2S-GalNAc6S GalNAc6S-UA2S-GalNAc4S6SGalNAc6S4S-UA-GalNAc GalNAc6S4S-UA-GalNAc4S GalNAc6S4S-UA-GalNAc6SGalNAc6S4S-UA-GalNAc4S6S GalNAc6S4S-UA2S-GalNAc GalNAc6S4S-UA2S-GalNAc4SGalNAc6S4S-UA2S-GalNAc6S GalNAc6S4S-UA2S-GalNAc4S6S

In further embodiments, one or more of the biomarkers used in anyprocess described herein includes one of the saturated biomarkers(glycan fragments) set forth in Table 11. In specific embodiments, thedisorder being diagnosed or otherwise analyzed is MPS IVB. In someembodiments, the first biomarker is a biomarker of Table 11 and thesecond biomarker is a biomarker of Table 8. In other embodiments, thefirst biomarker is a biomarker of Table 11 and the second biomarker is abiomarker of Table 9. In further or alternative embodiments, both thefirst and second biomarkers are from Table 11.

TABLE 11 KS derived CS Derived Glyco-lipid derived Gal GalNAcGal-GalNAc-Gal-Glu Gal-GlcNAc GalNAc4S Gal-GalNAc-Gal-Glu + 1Sialic acidGal-GlcNAc6S GalNAc-UA-GalNAc Gal-GalNAc-Gal-Glu + 2 Sialic acidsGal-GlcNAc-Gal GalNAc-UA-GalNAc4S Gal-GalNAc-Gal-Glu + 3 Sialic acidsGal-GlcNAc-Gal6S GalNAc-UA-GalNAc6S Gal-GlcNAc6S-GalGalNAc-UA-GalNAc4S6S Gal-GlcNAc6S-Gal6S GalNAc-UA2S-GalNAcGalNAc-UA2S-GalNAc4S GalNAc-UA2S-GalNAc6S GalNAc-UA2S-GalNAc4S6SGalNAc4S-UA-GalNAc GalNAc4S-UA-GalNAc4S GalNAc4S-UA-GalNAc6SGalNAc4S-UA-GalNAc4S6S GalNAc4S-UA2S-GalNAc GalNAc4S-UA2S-GalNAc4SGalNAc4S-UA2S-GalNAc6S GalNAc4S-UA2S-GalNAc4S6S

As used herein, IdoA and

are iduronic acid (e.g., α-L-iduronic acid) saccharide residues. As usedherein, GlcA and

are glucuronic acid (e.g., (3-L-glucuronic acid) saccharide residues. Asused herein,

are unsaturated uronic acids (UA), such as IdoA and GlcA. As usedherein, GlcN and

are glucosamine (e.g., 2-deoxy-2-amino-(3-D-glucopyranosyl) saccharideresidues. As used herein, GlcN(Ac)1 and

are a glucosamine (e.g., 2-deoxy-2-amino-(3-D-glucopyranosyl) saccharideresidue wherein the 2-amino group is acetylated. As used herein, Gal and◯ is a galactose saccharide residue. In various specific instances,iduronic acid, glucuronic acid, glucosamine, and/or galactose saccharideresidues are saturated at 4 and 5 carbons of the non-reducing endsaccharide residue, or are free of carbon-carbon unsaturation. In otherinstances, any one or more of the saccharide residues is unsaturated,e.g., at the 4 and 5 carbon positions of the saccharide residue at thenon-reducing end of an oligosaccharide provided herein. The symbolicnomenclature used herein follows the “Symbol and Text Nomenclature forRepresentation of Glycan Structure” as promulgated by the NomenclatureCommittee for the Consortium for Functional Glycomics, as amended onOctober 2007.

As an illustrative example of non-reducing end saccharide residues thatare saturated and unsaturated at the C4 and C5 positions, an L-iduronicacid (IdoA) residue that is saturated at the C4 and C5 positions has astructure as follows:

whereas an L-iduronic acid (IdoA) residue at the non-reducing end of theoligosaccharide that is unsaturated at the C4 and C5 positions may havea structure as follows:

or the like. Oligosaccharides having non-reducing end saccharideresidues that are saturated at the C4 and C5 position are referred toherein as “C4-C5 non-reducing end saturated oligosaccharides”.

Ratios of NRE Biomarkers:

In certain embodiments, a method provided for herein comprises comparinga first biomarker that is an NRE biomarker to a second biomarker that isa different NRE biomarker. In some embodiments, the first biomarker isspecific to a particular disease (e.g., MPS disease or other diseaseassociated with altered GAG synthesis or degradation). In certainembodiments, the second biomarker is an NRE biomarker that would not beexpected for the particular disease (e.g., according to the tablesprovided herein).

In some embodiments, a method described herein is used in concert withan ERT therapy. In some of such embodiments, the method is utilized tomonitor the efficacy of an ERT therapy.

In certain aspects, by examining the ratio of abundance of a diseasespecific NRE(s) to the NRE(s) that are generated after the action of anenzyme replacement therapy (ERT) in use, one can verify that the ERT isacting in the desired cellular compartment (the lysosome). In someinstances, this is important in order to ensure that a reduction in theglycan substrate in response to treatment reflects the beneficial actionof the ERT in the lysosome or the non-therapeutically beneficial actionof the enzyme outside of the lysosome. This is especially important intherapeutic approaches that require sampling fluids for biomarkeranalysis through the same port that the ERT was delivered—such asintrathecal delivery of ERT. If the ERT is acting outside of the cell(in blood, CSF, or in a sampling port) the subsequent lysosomal enzymeswill not efficiently degrade the resulting glycan. This leads to theelimination of the disease specific NRE and generation of a NREtypically associated with a different disease.

For example, in one specific embodiment, the disease being diagnosed orotherwise analyzed is MPS II. In some of such instances, the firstbiomarker is an MPS II disease specific NRE biomarker, such asIdoA2S-GlcN(+/−NS, +/−6S). If the ERT (2-sulfatase) acts in thelysosome, this NRE is 2-O desulfated producing IdoA-GlcN(+/−NS, +/−6S)which is rapidly eliminated by the subsequent lysosomal enzymes that arefunctional in MPS II patients. In contrast, if the ERT acts outside ofthe lysosome, the first biomarker (the MPS II NRE biomarker)[IdoA2S-GlcN(+/−NS, +/−6S)] is eliminated and the second biomarker(e.g., an MPS I NRE, such as [IdoA-GlcN(+/−NS, +/−6S)]) is generated(the extracellular action NRE, EANRE). In some instances, because theother lysosomal enzymes are not present in significant active quantitiesoutside of the lysosome, the MPS I markers are stable. In some cases theNREs can be converted to other NRE structures through the action ofother endogenous enzymes. In some aspects, using such techniques and bysimultaneous monitoring of the MPS II and MPS I NREs, the severity ofdisease and specific response to therapy are determined. Similar methodsfor the other MPS disorders, or any other disorder involving abnormalglycan accumulation, biosynthesis, and/or degradation are contemplatedherein.

In exemplary embodiments, the generation of an MPS I NRE biomarker in anMPS II patient after ERT treatment indicates that the ERT is noteffectively acting in the lysosome. In various aspects, the diseasespecific NRE and EANRE ratios that are relevant to each disease aredifferent for each disease class dependent on the NRE of the targetdisease and the relevant EANREs that are generated by the ERT. Inspecific exemplary embodiments methods described herein utilize thefollowing specific first and second biomarkers when utilized with thedenoted disease:

-   -   MPS I        -   Disease specific NREs (saturated fragments)            -   Disaccharides IdoA-GlcN(+/−NS, +/−6S)        -   EANREs            -   Mono and trisaccharides from the MPS IIIA and MPS IIIB                family    -   MPS II        -   Disease specific NREs (saturated fragments)            -   Disaccharides IdoA2S-GlcN(+/−NS, +/−6S)        -   EANREs            -   Disaccharides from the MPS I family    -   MPS IIIA        -   Disease specific NREs (saturated fragments)            -   Trisaccharides: GlcNS-GlcA/IdoA(+/−2S)-GlcN(+/−NS,                +/−6S)        -   EANREs            -   Disaccharides from the MPS IIIC family    -   MPS IIIB        -   Disease specific NREs (saturated fragments)            -   Trisaccharides: GlcNAc-GlcA/IdoA(+/−2S)-GlcN(+/−NS,                +/−6S)        -   EANREs            -   Disaccharides from the MPS I, II and VII families    -   MPS IIIC        -   Disease specific NREs (saturated fragments)            -   Trisaccharides: GlcN-GlcA/IdoA(+/−2S)-GlcN(+/−NS, +/−6S)        -   EANREs            -   Disaccharides from the MPS IIIB family    -   MPS IIID        -   Disease specific NREs (saturated fragments)            -   Trisaccharides:                GlcN(+/−NS)6S-GlcA/IdoA(+/−2S)-GlcN(+/−NS, +/−6S)        -   EANREs            -   Disaccharides from the MPS IIIA and IBB families    -   MPS IVA        -   Disease specific NREs (saturated fragments)            -   KS derived mono-, di, and trisaccharides: Gal6S,                Gal6S-GlcNAc(+/−6S), Gal6S-UA(+/−2S)-Gal(+/−6S)            -   CS derived mono-, di, and trisaccharides:                GalNAc6S(+/−4S), GalNAc6S(+/−4S)-UA(+/−2S)-GalNAc(+/−4S,                +/−6S)        -   EANREs            -   Mono and disacchares from the MPS IVB family    -   MPS VI        -   Disease specific NREs (saturated fragments)            -   CS derived mono and trisaccharides: GalNAc4S,                GalNAc4S-UA(+/−2S)-GalNAc(+/−4S, +/−6S)        -   EANREs            -   NREs from hexosaminindase deficiencies    -   MPS VII        -   Disease specific NREs (saturated fragments)            -   Disaccharides: GlcA-GlcN(+/−NS, +/−6S)        -   EANREs            -   Disaccharides from the MPS IIIA, IIIB, and IIID families

Ratios of NRE and Non-NRE Biomarkers:

In certain embodiments, a method provided for herein comprises comparinga first biomarker that is an NRE biomarker to a second biomarker that isa non-NRE biomarker (e.g., a reducing end or internal glycan residualbiomarker). In some embodiments, the first biomarker is specific to aparticular disease (e.g., MPS disease). In specific embodiments, thesecond biomarker is an internal biomarker (e.g., from Table 9). In morespecific embodiments, such methods are utilized in combination with atherapy for the treatment of a disorder associated with abnormal glycanbiosynthesis, degradation, and/or accumulation.

In certain embodiments, the second biomarker is a non-NRE marker (e.g.,rather than an EANRE discussed above). Because the non-therapeuticaction of the ERT only eliminates the specific NRE structure, but doesnot reduce the level of the accumulating glycan, ratios of the diseasespecific NRE to other non-NRE structures can also be used to determinethe site of action of the ERT.

In an exemplary embodiment, wherein the disease being diagnosed orotherwise analyzed is MPS II, a disease specific NRE isIdoA2S-GlcN(+/−NS, +/−6S). If the ERT (2-sulfatase) acts in thelysosome, this NRE is 2-O desulfated producing a GAG fragment thatterminates with IdoA-GlcN(+/−NS, +/−6S). That fragment is rapidlyeliminated by the subsequent lysosomal enzymes that are functional inMPS II patients. In contrast, if the ERT acts outside of the lysosome,the abundance of internal HS fragments liberated by lyase digestionremain constant. Therefore, by simultaneous monitoring of the MPS II andinternal HS derived structures such as ΔUA-GlcNAc or ΔUA-GlcNS the truelysosomal activity of the treatment can be measured.

In some embodiments, a method described herein is utilized to determinethe severity of a disease described herein. In some of such embodiments,the ratios of abundance of different biomarkers (e.g., NRE biomarkers)may be analyzed and utilized to determine disease severity and/orresponse to therapy.

For example, each MPS class has a number of specific NRE structures thataccumulate. In some embodiments, the ratio of the different specificstructures change as the disease severity changes. For example in MPS IIthere are a number of HS derived NREs (IdoA2S-GlcNS, IdoA2S-GlcNS6S,IdoA2S-GlcNAc, IdoA2S-GlcNAc6S) and CS/DS derived NREs (IdoA2S-GalNAc4S,IdoA2S-GalNAc6S, IdoA2S-GalNAc, IdoA2S-GalNAc4S6S) which are found indifferent abundance depending on the severity of disease. By monitoringthe ratio of these distinct NRE structures, information about theseverity of the disease and response to therapy can be obtained.

In some embodiments, a method described herein is utilized to indentifydisease. In specific embodiments, ratios of NRE biomarkers or InternalGAG lyase generated biomarkers are utilized in such methods. In specificinstances, the ratios of abundance of different NRE and internalunsaturated saccharides generated after lyase digestion can be used toindicate the presence of human disease.

In specific embodiments, a method described herein is utilized toidentify Schneckenbecken dysplasia (which leads to reduced UDP sugardonors which alters ratios of glycosminoglycan lyase generated fragmentsoriginating from HS, CS, and DS). In other exemplary embodiments, adeficiency in an enzyme required for the 2-O sulfation of heparansulfate can be identified by examining the ratio of unsaturated 2-Osulfated disaccharides (generated by lyase digestion) to non-2-Osulfated disaccharides. In some instances, a reduction in this ratioindicates the disruption of heparan sulfate 2-O sulfation and thepresence of human disease. In further exemplary embodiments, adeficiency in the biosynthesis of 4-O sulfated chondroitin and dermatansulfate can be identified by examining the ratio of unsaturated 4-Osulfated disaccharides (generated after lyase digestion to non-4-Osulfated disaccharides). In some instances, a reduction in this ratioindicates the disruption of chondroitin sulfate 4-O sulfation and thepresence of human disease. In still further exemplary embodiments, adeficiency in PAPs (3-prime-phosphoadenosine 5-prime-phosphosulfate)synthesis or transport can be identified in changes in the ratio ofabundance or ratios of lyase generated glycosaminoglycan fragments.

Disorders:

In certain embodiments, a disorder associated with abnormal glycanaccumulation includes a disorder associated therewith is caused by anabnormally functioning glycan degradation enzyme. In variousembodiments, the abnormally functioning glycan degradation enzymefunctions abnormally as a result of being present in an abnormally lowamount, functioning improperly, or a combination thereof. For example,an abnormally functioning glycan degradation enzyme functions abnormallyas a result of being present in an amount of less than 50%, less than40%, less than 30%, less than 20%, less than 10%, or less than 5% thanis present in an individual with normal amounts of the glycandegradation enzyme (e.g., an individual in a non-diseased, normal, orwild type state). In further or alternative embodiments, abnormallyfunctioning glycan degradation enzymes are present in a normal amount,but do not function properly in degrading glycans. For example, suchenzymes may be have amino acid substitutions in the sequences thereofthat reduce or eliminate the glycan degradative properties of theenzyme.

MPS I is a human genetic disease caused by a deficiency in the lysosomalenzyme L-iduronidase. This enzyme is required in the lysosome to degradeglycans that contain iduronic acid. Due to this enzymatic deficiency,glycans with an iduronic acid on the non-reducing end accumulate to highlevels (including heparan sulfate and dermatan sulfate). In certainembodiments, using the method described herein, MPS I is diagnosed in anindividual from a biological sample taken therefrom. For example, insome embodiments, a biological sample is optionally placed into adefined MW cut off spin column (retains large molecules when spun),optionally washed (e.g., with water or buffer) to remove freemonosaccharides, then treated with an iduronidase (e.g., to liberate aglycan residual compound iduronic acid). In certain embodiments, afterincubation, the liberated iduronic acid is isolated, e.g., by washingthe free monosaccharide through the defined MW cut off membrane (orother methods). In some of such embodiments, the monosaccharide would bein the flow through. The isolated monosaccharide solution is optionallydried or otherwise treated to concentrate the sample and subsequentlyanalyzed for iduronic acid content by any suitable analytical technique(e.g., HPLC, MS, GC, or the like with or without chemical or enzymaticderivatization before detection). This method can be used to detect MPSI disease, measure disease severity, or to measure response to therapy.

MPS II is a human genetic disease caused by a deficiency in thelysosomal enzyme 2-sulfatase. This enzyme is required in the lysosome todegrade glycans that contain 2-O sulfated uronic acids. Due to thisenzymatic deficiency, glycans with a 2-sulfated uronic acid on thenon-reducing end accumulate to high levels (including heparan sulfateand dermatan sulfate). In certain embodiments, using the methoddescribed herein, MPS II is diagnosed in an individual from a biologicalsample taken therefrom. For example, in some embodiments, a biologicalsample is optionally placed in to a defined MW cut off spin column(retains large molecules when spun), optionally washed (e.g., with 1 ormore volumes of water or buffer to remove free sulfate), and treatedwith a 2-sulfatase (e.g., to liberate a glycan residual compoundsulfate). In some embodiments, after incubation, the liberated sulfateis optionally isolated by washing the free monosaccharide (e.g., througha defined MW cut off membrane or by any other suitable method). In someof such embodiments, the free sulfate is in the flow through. In certainembodiments, the resulting isolated solution is optionally dried orotherwise treated to concentrate the sample and subsequently analyzedfor sulfate content by any suitable analytical technique (e.g., HPLC,MS, GC, pH detection, or the like with or without chemical or enzymaticderivatization before detection). This method can be used to detect MPSII disease, measure disease severity, or to measure response to therapy.In other exemplary embodiments, following treatment with a 2-sulfatase,the resulting 2-O desulfated non-reducing end uronic acid residues isoptionally liberated with an iduronidase or glucuronidase. In some ofsuch embodiments, the resulting liberated monosaccharide is optionallyisolated, e.g., by washing free monosaccharide (e.g., through thedefined MW cut off membrane or any other suitable method). In some ofsuch embodiments, free iduronic or glucuronic acid is in the flowthrough. In certain embodiments, the resulting isolated solution isoptionally dried or otherwise treated to concentrate the sample andsubsequently analyzed for monosaccharide content by any suitableanalytical technique (e.g., HPLC, MS, GC, or the like with or withoutchemical or enzymatic derivatization before detection). This method canbe used to detect MPS II disease, measure disease severity, or tomeasure response to therapy.

MPS IIIA is a human genetic disease caused by a deficiency in thelysosomal enzyme N-sulfatase. This enzyme is required in the lysosome todegrade glycans that contain N-sulfated glucosamine residues. Due tothis enzymatic deficiency, glycans with N-sulfated glucosamine residueson the non-reducing end accumulate to high levels (including heparansulfate). In certain embodiments, using the method described herein, MPSIIIA is diagnosed in an individual from a biological sample takentherefrom. For example, in some embodiments, a biological sample isoptionally placed in to a defined MW cut off spin column (retains largemolecules when spun), optionally washed (e.g., with 1 or more volumes ofwater or buffer) to remove free sulfate, and treated with anN-sulfatase. In certain embodiments, after incubation, the liberatedsulfate is optionally isolated, e.g., by washing the free monosaccharide(such as through a defined MW cut off membrane or any other suitablemethod). In some of such embodiments, free sulfate for detection and/orquantitation in the flow through. In certain embodiments, the resultingisolated solution is optionally dried or otherwise treated toconcentrate the sample and subsequently analyzed for sulfate content byany suitable analytical technique (e.g., HPLC, MS, GC, pH detection, orthe like with or without chemical or enzymatic derivatization beforedetection). This method can be used to detect MPS IIIA disease, measuredisease severity, or to measure response to therapy. In further oralternative embodiments, following treatment with an N-sulfatase, theresulting N-desulfated non-reducing end glucosamine residues isoptionally liberated with a hexosaminidase. In some of such embodiments,liberated monosaccharide is optionally isolated (e.g., by washing thefree monosaccharide, such as through the defined MW cut off membrane orany other suitable method). In some of such embodiments, freeglucosamine for detection and/or quantitation is present in the flowthrough. In certain embodiments, the resulting isolated solution isoptionally dried or otherwise treated to concentrate the sample andsubsequently analyzed for monosaccharide content by any suitableanalytical technique (e.g., HPLC, MS, GC, or the like with or withoutchemical or enzymatic derivatization before detection). This method canbe used to detect MPS IIIA disease, measure disease severity, or tomeasure response to therapy.

As discussed above, in certain embodiments, using the method describedherein, MPS IIIA is diagnosed in an individual from a biological sampletaken therefrom. For example, in some embodiments, a biological sampleis optionally placed in to a defined MW cut off spin column (retainslarge molecules when spun), optionally washed (e.g., with 1 or morevolumes of water or buffer) to remove free monosaccharide, and treatedwith an N-sulfo glucosaminidase such as a heparin lyase. In someembodiments, liberated sulfated monosaccharide is optionally isolated,e.g., by washing the free monosaccharide (such as through the defined MWcut off membrane or by any other suitable method). In some of suchembodiments, free N-sulfated glucosamine for detection and/orquantitation is present in the flow through. In certain embodiments, theresulting isolated solution is optionally dried or otherwise treated toconcentrate the sample and subsequently analyzed for monosaccharidecontent by any suitable analytical technique (e.g., HPLC, MS, GC, or thelike with or without chemical or enzymatic derivatization beforedetection). This method can be used to detect MPS IIIA disease, measuredisease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method describedherein, MPS IIIA is diagnosed in an individual from a biological sampletaken therefrom. For example, in some embodiments, a biological sampleis optionally placed in to a defined MW cut off spin column (retainslarge molecules when spun), optionally washed (e.g., with 1 or morevolumes of water or buffer) to remove free monosaccharide, and treatedwith an N-sulfatase. In certain embodiments, the resulting glycan issubsequently treated such that the N-desulfated non-reducing endglucosamine residues is acetylated (e.g., with an N-acetyl transferase)and subsequently liberated with a hexosaminidase. In some of suchembodiments, the resulting liberated monosaccharide is optionallyisolated, e.g., by washing the free monosaccharide (e.g., through adefined MW cut off membrane or any other suitable methods). In some ofsuch embodiments, free N-acetyl glucosamine for detection and/orquantitation is present in the flow through. In certain embodiments, theresulting isolated composition is optionally dried or otherwise treatedto concentrate the sample and subsequently analyzed for monosaccharidecontent by any suitable analytical technique (e.g., HPLC, MS, GC, or thelike with or without chemical or enzymatic derivatization beforedetection). This method can be used to detect MPS IIIA disease, measuredisease severity, or to measure response to therapy.

MPS IIIB is a human genetic disease caused by a deficiency in the enzymeN-acetyl glucosaminidase. This enzyme is required in the lysosome todegrade glycans that contain N-acetyl glucosamine residues. Due to thisenzymatic deficiency, glycans with a N-acetyl glucosamine residue on thenon-reducing end accumulate to high levels (including heparan sulfate).In certain embodiments, using the method described herein, MPS IIIB isdiagnosed in an individual from a biological sample taken therefrom. Forexample, in some embodiments, a biological sample is optionally placedin to a defined MW cut off spin column (retains large molecules whenspun), optionally washed (e.g., with 1 or more volumes of water orbuffer to remove free N-acetyl glucosamine), and treated with a-acetylglucosaminidase or a heparin lyase (e.g., to liberate a glycan residualcompound N-acetyl glucosamine). In some embodiments, after incubation,the liberated N-acetyl glucosamine is optionally isolated by washing thefree monosaccharide (e.g., through a defined MW cut off membrane or byany other suitable method). In some of such embodiments, the freemonosaccharide is in the flow through. In certain embodiments, theresulting isolated solution is optionally dried or otherwise treated toconcentrate the sample and subsequently analyzed for monosaccharidecontent by any suitable analytical technique (e.g., HPLC, MS, GC, pHdetection, or the like with or without chemical or enzymaticderivatization before detection). This method can be used to detect MPSIIIB disease, measure disease severity, or to measure response totherapy.

As discussed above, in certain embodiments, using the method describedherein, MPS IIIA is diagnosed in an individual from a biological sampletaken therefrom. For example, in some embodiments, a biological sampleis optionally placed in to a defined MW cut off spin column (retainslarge molecules when spun), optionally washed (e.g., with 1 or morevolumes of water or buffer) to remove free acetate, and treated with adeacetylase. The liberated acetate is optionally isolated, e.g., bywashing the free acetate (such as through the defined MW cut offmembrane or any other suitable method). In some of such embodiments, thefree acetate for detection and/or quantitation is present the flowthrough. In some embodiments, the resulting isolated solution isoptionally dried or otherwise treated to concentrate the sample andsubsequently analyzed for acetate content by any suitable analyticaltechnique (e.g., HPLC, MS, GC, pH detection, or the like with or withoutchemical or enzymatic derivatization before detection). This method canbe used to detect MPS IIIB disease, measure disease severity, or tomeasure response to therapy.

MPS IIIC is a human genetic disease caused by a deficiency in the enzymeN-acetyltransferase. This enzyme is required in the lysosome to degradeglycans that contain glucosamine residues. Due to this enzymaticdeficiency, glycans with a glucosamine residue on the non-reducing endaccumulate to high levels (including heparan sulfate). In certainembodiments, using the method described herein, MPS IIIC is diagnosed inan individual from a biological sample taken therefrom. For example, insome embodiments, a biological sample is optionally placed in to adefined MW cut off spin column (retains large molecules when spun),optionally washed (e.g., with 1 or more volumes of water or buffer toremove free glucosamine), and treated with a hexosaminidase or heparinlyase (e.g., to liberate a glycan residual compound glucosamine). Insome embodiments, after incubation, the liberated glucosamine isoptionally isolated by washing the free glucosamine (e.g., through adefined MW cut off membrane or by any other suitable method). In some ofsuch embodiments, the free glucosamine for detection and/or quantitationis present in the flow through. In certain embodiments, the resultingisolated solution is optionally dried or otherwise treated toconcentrate the sample and subsequently analyzed for monosaccharidecontent by any suitable analytical technique (e.g., HPLC, MS, GC, pHdetection, or the like with or without chemical or enzymaticderivatization before detection). This method can be used to detect MPSIIIC disease, measure disease severity, or to measure response totherapy.

As discussed above, in certain embodiments, using the method describedherein, MPS IIIC is diagnosed in an individual from a biological sampletaken therefrom. For example, in some embodiments, a biological sampleis optionally placed in to a defined MW cut off spin column (retainslarge molecules when spun), optionally washed (e.g., with 1 or morevolumes of water or buffer to remove free glucosamine and/or N-acetylglucosamine), and treated with a glucosamine N-acetyltransferasefollowed by a hexosaminidase (e.g., to liberate a glycan residualcompound N-acetyl glucosamine). In some embodiments, after incubation,the liberated N-acetyl glucosamine is optionally isolated by washing thefree N-acetyl glucosamine (e.g., through a defined MW cut off membraneor by any other suitable method). In some of such embodiments, the freeN-acetyl glucosamine for detection and/or quantitation is present in theflow through. In certain embodiments, the resulting isolated solution isoptionally dried or otherwise treated to concentrate the sample andsubsequently analyzed for monosaccharide content by any suitableanalytical technique (e.g., HPLC, MS, GC, pH detection, or the like withor without chemical or enzymatic derivatization before detection). Thismethod can be used to detect MPS IIIC disease, measure disease severity,or to measure response to therapy.

MPS IIID is a human genetic disease caused by a deficiency in the enzymeglucosamine 6-O sulfatase. This enzyme is required in the lysosome todegrade glycans that contain 6-O-sulfated glucosamine residues. Due tothis enzymatic deficiency, glycans with a 6-O-sulfated N-acetylglucosamine residue on the non-reducing end accumulate to high levels(including heparan sulfate). In certain embodiments, using the methoddescribed herein, MPS IIIC is diagnosed in an individual from abiological sample taken therefrom. For example, in some embodiments, abiological sample is optionally placed in to a defined MW cut off spincolumn (retains large molecules when spun), optionally washed (e.g.,with 1 or more volumes of water or buffer to remove free sulfate), andtreated with a 6-O-sulfatase (e.g., to liberate a glycan residualcompound sulfate). In some embodiments, after incubation, the liberatedsulfate is optionally isolated by washing the free sulfate (e.g.,through a defined MW cut off membrane or by any other suitable method).In some of such embodiments, the free sulfate for detection and/orquantitation is present in the flow through. In certain embodiments, theresulting isolated solution is optionally dried or otherwise treated toconcentrate the sample and subsequently analyzed for sulfate content byany suitable analytical technique (e.g., HPLC, MS, GC, pH detection, orthe like with or without chemical or enzymatic derivatization beforedetection). This method can be used to detect MPS IIID disease, measuredisease severity, or to measure response to therapy.

As discussed above, in certain embodiments, using the method describedherein, MPS IIID is diagnosed in an individual from a biological sampletaken therefrom. For example, in some embodiments, a biological sampleis optionally placed in to a defined MW cut off spin column (retainslarge molecules when spun), optionally washed (e.g., with 1 or morevolumes of water or buffer to remove free sulfate and/or N-acetylglucosamine), and treated with a 6-O-sulfatase and a hexosaminidase(e.g., to liberate a glycan residual compound N-acetyl glucosamine). Insome embodiments, after incubation, the liberated N-acetyl glucosamineis optionally isolated by washing the free N-acetyl glucosamine (e.g.,through a defined MW cut off membrane or by any other suitable method).In some of such embodiments, the free monosaccharide for detectionand/or quantitation is present in the flow through. In certainembodiments, the resulting isolated solution is optionally dried orotherwise treated to concentrate the sample and subsequently analyzedfor monosaccharide content by any suitable analytical technique (e.g.,HPLC, MS, GC, or the like with or without chemical or enzymaticderivatization before detection). This method can be used to detect MPSIIID disease, measure disease severity, or to measure response totherapy.

As discussed above, in certain embodiments, using the method describedherein, MPS IIID is diagnosed in an individual from a biological sampletaken therefrom. For example, in some embodiments, a biological sampleis optionally placed in to a defined MW cut off spin column (retainslarge molecules when spun), optionally washed (e.g., with 1 or morevolumes of water or buffer to remove free sulfate and/or N-acetylglucosamine 6-O sulfate), and treated with a hexosaminidase or heparinlyase (e.g., to liberate a glycan residual compound N-acetyl glucosamine6-O sulfate). In some embodiments, after incubation, the liberatedN-acetyl glucosamine 6-O sulfate is optionally isolated by washing thefree N-acetyl glucosamine 6-O sulfate (e.g., through a defined MW cutoff membrane or by any other suitable method). In some of suchembodiments, the free monosaccharide for detection and/or quantitationis present in the flow through. In certain embodiments, the resultingisolated solution is optionally dried or otherwise treated toconcentrate the sample and subsequently analyzed for monosaccharidecontent by any suitable analytical technique (e.g., HPLC, MS, GC, pHdetection, or the like with or without chemical or enzymaticderivatization before detection). This method can be used to detect MPSIIID disease, measure disease severity, or to measure response totherapy.

MPS IVA is a human genetic disease caused by a deficiency in the enzymelysosomal enzyme galactose/N-acetyl galactosamine 6-O sulfatase. Thisenzyme is required in the lysosome to degrade glycans that contain6-O-sulfated galactose and 6-O sulfated N-acetyl galactosamine residues.Due to this enzymatic deficiency, glycans with 6-O-sulfated galactoseand 6-O sulfated N-acetyl galactosamine residues on the non-reducing endaccumulate to high levels (including chondroitin and keratan sulfate).In certain embodiments, using the method described herein, MPS IVA isdiagnosed in an individual from a biological sample taken therefrom. Forexample, in some embodiments, a biological sample is optionally placedin to a defined MW cut off spin column (retains large molecules whenspun), optionally washed (e.g., with 1 or more volumes of water orbuffer to remove free monosaccharide), and treated with a galactose6-O-sulfatase and/or an N-acetyl galactosamine 6-O sulfatase and agalactosidase and/or hexosaminidase (e.g., to liberate a glycan residualcompound Gal and/or GalNAc). In some embodiments, after incubation, theliberated monosaccharide is optionally isolated by washing the freemonosaccharide (e.g., through a defined MW cut off membrane or by anyother suitable method). In some of such embodiments, the freemonosaccharide for detection and/or quantitation is present in the flowthrough. In certain embodiments, the resulting isolated solution isoptionally dried or otherwise treated to concentrate the sample andsubsequently analyzed for monosaccharide content by any suitableanalytical technique (e.g., HPLC, MS, GC, or the like with or withoutchemical or enzymatic derivatization before detection). This method canbe used to detect MPS IVA disease, measure disease severity, or tomeasure response to therapy.

As discussed above, in certain embodiments, using the method describedherein, MPS IVA is diagnosed in an individual from a biological sampletaken therefrom. For example, in some embodiments, a biological sampleis optionally placed in to a defined MW cut off spin column (retainslarge molecules when spun), optionally washed (e.g., with 1 or morevolumes of water or buffer to remove free sulfate), and treated with a6-O-sulfatase capable of desulfating 6-O-sulfated galactose and/or 6-Osulfated N-acetyl galactosamine residues (e.g., to liberate a glycanresidual compound sulfate). In some embodiments, after incubation, theliberated sulfate is optionally isolated by washing the free sulfate(e.g., through a defined MW cut off membrane or by any other suitablemethod). In some of such embodiments, the free sulfate for detectionand/or quantitation is present in the flow through. In certainembodiments, the resulting isolated solution is optionally dried orotherwise treated to concentrate the sample and subsequently analyzedfor sulfate content by any suitable analytical technique (e.g., HPLC,MS, GC, pH detection, or the like with or without chemical or enzymaticderivatization before detection). This method can be used to detect MPSIVA disease, measure disease severity, or to measure response totherapy.

MPS IVB is a human genetic disease caused by a deficiency in the enzymelysosomal β-galactosidase. This enzyme is required in the lysosome todegrade glycans that contain galactose residues. Due to this enzymaticdeficiency, glycans with β-galactose residues on the non-reducing endaccumulate to high levels (including keratan sulfate and other glycans).In certain embodiments, using the method described herein, MPS IVB isdiagnosed in an individual from a biological sample taken therefrom. Forexample, in some embodiments, a biological sample is optionally placedin to a defined MW cut off spin column (retains large molecules whenspun), optionally washed (e.g., with 1 or more volumes of water orbuffer to remove free monosaccharide), and treated with a galactosidase(e.g., to liberate a glycan residual compound Gal). In some embodiments,after incubation, the liberated monosaccharide is optionally isolated bywashing the free monosaccharide (e.g., through a defined MW cut offmembrane or by any other suitable method). In some of such embodiments,the free monosaccharide for detection and/or quantitation is present inthe flow through. In certain embodiments, the resulting isolatedsolution is optionally dried or otherwise treated to concentrate thesample and subsequently analyzed for monosaccharide content by anysuitable analytical technique (e.g., HPLC, MS, GC, or the like with orwithout chemical or enzymatic derivatization before detection). Thismethod can be used to detect MPS IVB disease, measure disease severity,or to measure response to therapy.

MPS VI is a human genetic disease caused by a deficiency in the enzyme4-O sulfatase that desulfates N-acetyl galactosamine. This enzyme isrequired in the lysosome to degrade glycans that contain 4-O-sulfatedN-acetyl galactosamine residues. Due to this enzymatic deficiency,glycans with 4-O-sulfated N-acetyl galactosamine residues on thenon-reducing end accumulate to high levels (including chondroitinsulfate). In certain embodiments, using the method described herein, MPSVI is diagnosed in an individual from a biological sample takentherefrom. For example, in some embodiments, a biological sample isoptionally placed in to a defined MW cut off spin column (retains largemolecules when spun), optionally washed (e.g., with 1 or more volumes ofwater or buffer to remove free sulfate), and treated with a4-O-sulfatase that can desulfate 4-O-sulfated N-acetyl galactosamineresidues (e.g., to liberate a glycan residual compound sulfate). In someembodiments, after incubation, the liberated sulfate is optionallyisolated by washing the free sulfate (e.g., through a defined MW cut offmembrane or by any other suitable method). In some of such embodiments,the free sulfate for detection and/or quantitation is present in theflow through. In certain embodiments, the resulting isolated solution isoptionally dried or otherwise treated to concentrate the sample andsubsequently analyzed for sulfate content by any suitable analyticaltechnique (e.g., HPLC, MS, GC, pH detection, or the like with or withoutchemical or enzymatic derivatization before detection). This method canbe used to detect MPS VI disease, measure disease severity, or tomeasure response to therapy.

As discussed above, in certain embodiments, using the method describedherein, MPS VI is diagnosed in an individual from a biological sampletaken therefrom. For example, in some embodiments, a biological sampleis optionally placed in to a defined MW cut off spin column (retainslarge molecules when spun), optionally washed (e.g., with 1 or morevolumes of water or buffer to remove free N-acetyl galactosamine), andtreated with a 4-O-sulfatase that is capable of desulfating 4-O-sulfatedN-acetyl galactosamine residues then treated with a hexosaminidase(e.g., to liberate a glycan residual compound N-acetyl galactosamine).In some embodiments, after incubation, the liberated N-acetylgalactosamine is optionally isolated by washing the free monosaccharide(e.g., through a defined MW cut off membrane or by any other suitablemethod). In some of such embodiments, the free monosaccharide fordetection and/or quantitation is present in the flow through. In certainembodiments, the resulting isolated solution is optionally dried orotherwise treated to concentrate the sample and subsequently analyzedfor monosaccharide content by any suitable analytical technique (e.g.,HPLC, MS, GC, or the like with or without chemical or enzymaticderivatization before detection). This method can be used to detect MPSVI disease, measure disease severity, or to measure response to therapy.

MPS VII is a human genetic disease caused by a deficiency in thelysosomal enzyme beta-glucuronidase. This enzyme is required in thelysosome to degrade glycans that contain glucuronic acid residues. Dueto this enzymatic deficiency, glycans with glucuronic acid residues onthe non-reducing end accumulate to high levels (including chondroitinsulfate, heparan sulfate and others). In certain embodiments, using themethod described herein, MPS VII is diagnosed in an individual from abiological sample taken therefrom. For example, in some embodiments, abiological sample is optionally placed in to a defined MW cut off spincolumn (retains large molecules when spun), optionally washed (e.g.,with 1 or more volumes of water or buffer to remove free glucuronicacid), and treated with a glucuronidase (e.g., to liberate a glycanresidual compound glucuronic acid). In some embodiments, afterincubation, the liberated monosaccharide is optionally isolated bywashing the free monosaccharide (e.g., through a defined MW cut offmembrane or by any other suitable method). In some of such embodiments,the free monosaccharide for detection and/or quantitation is present inthe flow through. In certain embodiments, the resulting isolatedsolution is optionally dried or otherwise treated to concentrate thesample and subsequently analyzed for monosaccharide content by anysuitable analytical technique (e.g., HPLC, MS, GC, pH detection, or thelike with or without chemical or enzymatic derivatization beforedetection). This method can be used to detect MPS VII disease, measuredisease severity, or to measure response to therapy.

Methods described herein can also be used to define the relativepresence of different glycan classes.

Fabry Disease is a human genetic disease caused by a deficiency in thelysosomal α-galactosidase. Due to this enzymatic deficiency, glycanswith non-reducing end terminal α-galactose residues are abundant. Incertain embodiments, using the method described herein, Fabry Disease isdiagnosed in an individual from a biological sample taken therefrom. Forexample, in some embodiments, a biological sample is optionally placedin to a defined MW cut off spin column (retains large molecules whenspun), optionally washed (e.g., with 1 or more volumes of water orbuffer to remove free monosaccharide), and treated with a galactosidasethat is capable of liberating a non-reducing end monosaccharide (e.g.,to liberate a glycan residual compound). In some embodiments, afterincubation, the liberated glycan residual compound is optionallyisolated by washing the free glycan residual compound (e.g., through adefined MW cut off membrane or by any other suitable method). In some ofsuch embodiments, the free glycan residual compound for detection and/orquantitation is present in the flow through. In certain embodiments, theresulting isolated solution is optionally dried or otherwise treated toconcentrate the sample and subsequently analyzed for glycan residualcompound content by any suitable analytical technique (e.g., HPLC, MS,GC, pH detection, or the like with or without chemical or enzymaticderivatization before detection). This method can be used to detectFabry Disease, measure disease severity, or to measure response totherapy.

In some embodiments, as described in Table 1, other enzymes andprocesses are optionally utilized to diagnose other lysosomal storagediseases (LSDs). As described in the table, the appropriate enzyme(s)can be selected as appropriate for the specific disease.

Oncology—Melanoma and Neuroblastoma via Sialic Acid

A hallmark of cancer is altered glycosylation. The changes inglycosylation are a reflection of changes in enzymes and factors thatregulate the biosynthesis, turnover, presentation, stability,solubility, and degradation of glycans. Many of these changes result inglycans being produced that have altered structures. The methodsdescribed here are utilized in various embodiments to evaluate thosestructural changes (e.g., measure abnormal glycan accumulation) that arepresent on the non-reducing end of the glycans present in individualssuffering from a cancerous disease.

Some examples of cancerous diseases suitable for diagnosis and/ormonitoring therapy according to methods described herein include, by wayof non-limiting example, melanoma and neuroblastoma. In some instances,such cancers have alterations in the biosynthesis, turnover,presentation, stability, solubility, or degradation of gangliosides. Insome instances, these sialic acid modified glycolipids are detectedand/or otherwise characterized or analyzed in a biological sample (e.g.,serum) of patients with these tumor types. In some embodiments, theabundance of the heterogeneous population of gangliosides is quantifiedto measuring sialic acid or other glycan residual released fromgangliosides in the blood.

Due to this enzymatic alteration, gangliosides and other glycans arepresent in the body at high levels. In certain embodiments, using themethod described herein, cancer (e.g., melanoma or neuroblastoma) isdiagnosed in an individual from a biological sample taken therefrom. Forexample, in some embodiments, a biological sample is optionally placedin to a defined MW cut off spin column (retains large molecules whenspun), optionally washed (e.g., with 1 or more volumes of water orbuffer to remove free sialic acid), and treated with a sialidase thatcan liberate sialic acid (e.g., to liberate a glycan residual compoundsialic acid). In some embodiments, after incubation, the liberatedsialic acid is optionally isolated by washing the free sialic acid(e.g., through a defined MW cut off membrane or by any other suitablemethod). In some of such embodiments, the free sialic acid for detectionand/or quantitation is present in the flow through. In certainembodiments, the resulting isolated solution is optionally dried orotherwise treated to concentrate the sample and subsequently analyzedfor sialic acid content by any suitable analytical technique (e.g.,HPLC, MS, GC, pH detection, or the like with or without chemical orenzymatic derivatization before detection). This method can be used todetect cancer (e.g., melanoma or neuroblastoma) disease, measure diseaseseverity, or to measure response to therapy.

Oncology—Myeloma Via Heparan Sulfate Nonreducing Ends

An example of a human cancer that is diagnosed and/or monitoredaccording to the methods described herein (i.e., by analyzing with sucha method the altered degradation of a glycan) is multiple myeloma. Incertain instances, multiple myeloma commonly produces heparanase.Heparanase is an endoglycosidase that cleaved heparan sulfate intosmaller fragments, exposing novel non-reducing end structures. Incertain embodiments described herein, the presence of these novelnon-reducing end structures are detected using any method describedherein (e.g., by incubating a biological sample with variousglycosidases or sulfatases to detect the presence of novel glycannon-reducing ends).

Due to this enzymatic alteration, glycans (including heparan sulfate andothers) are present in the body at high levels. In certain embodiments,using the method described herein, cancer (e.g., multiple myeloma) isdiagnosed in an individual from a biological sample taken therefrom. Forexample, in some embodiments, a biological sample is optionally placedin to a defined MW cut off spin column (retains large molecules whenspun), optionally washed (e.g., with 1 or more volumes of water orbuffer to remove free monosaccharides and/or sulfate), and treated witha sulfatase, iduronidase, glucuronidase, hexosaminidase, or lyase thatis capable of liberating a non-reducing end monosaccharide or sulfate.In some embodiments, after incubation, the liberated glycan residualcompound is optionally isolated by washing the free glycan residualcompound (e.g., through a defined MW cut off membrane or by any othersuitable method). In some of such embodiments, the free glycan residualcompound for detection and/or quantitation is present in the flowthrough. In certain embodiments, the resulting isolated solution isoptionally dried or otherwise treated to concentrate the sample andsubsequently analyzed for glycan residual compound content by anysuitable analytical technique (e.g., HPLC, MS, GC, pH detection, or thelike with or without chemical or enzymatic derivatization beforedetection). This method can be used to detect cancer (e.g., multiplemyeloma) disease, measure disease severity, or to measure response totherapy.

Oncology—Adenocarcinoma

Adenocarcinoma is associated with changes in glycosylation includingincreased sialylation and fucosylation. The described method can be usedto measure disease by analyzing glycans (total or purified or enrichedfor specific glycan classes) from a patient for the amount ofnonreducing end terminal sialic acid or fucose, by measuring the releaseof these glycan residuals after treatment with a sialidase orfucosidase.

Other Applications

As described in Tables 12-15, various diseases associated with changesin glycosylation are optionally diagnosed and/or monitored according tomethods described herein. Various disorders include, by way ofnon-limiting example, lysosomal storage disease, cancer, neurologicaldisease (dementia, Alzheimer's, etc), liver disease, bone disease,infectious diseases, and the like.

Provided herein are methods of diagnosing individuals (including, e.g.,a disease state or the severity of a disease states) with a lysosomalstorage disease (LSD) or methods of monitoring the treatment of alysosomal storage disease (LSD). Provided in Table 10 are specificembodiments of disease that are optionally diagnosed and/or monitoredaccording to various embodiments described herein. Table 12 alsoillustrates various non-limiting embodiments of specific enzyme(s) thatare optionally utilized to treat a biological sample from an individualsuffering from or suspected (e.g., through a pre- or preliminaryscreening process) of suffering from an LSD. Moreover, Table 12 furtherillustrates various glycan residual compounds that are liberated invarious embodiments described herein, such liberated glycan residualcompounds optionally being detected and/or measured in order to diagnoseand/or monitor a lysosomal storage disease (LSD).

TABLE 12 Exemplary LSD Uses Primary Secondary Glycan Non-ReducingReleasing Releasing Residual Disease End Structure Enzyme EnzymeCompound MPS I IdoA iduronidase IdoA MPS II IdoA-2-O sufate 2-sulfataseSulfate and GlcA-2-O sufate MPS II IdoA-2-O sufate 2-sulfataseIduronidase IdoA and/or and GlcA-2-O sufate and/or GlcA glucuronidaseMPS IIIA GlcN—N-sulfate N-sulfatase Sulfate MPS IIIA GlcN—N-sulfateN-sulfatase hexosaminidase GlcN MPS IIIA GlcN—N-sulfate N-sulfataseHeparin lyase GlcN MPS IIIA GlcN—N-sulfate N-sulfatase N-acetyl GlcNActransferase and hexosaminidase MPS IIIA GlcN—N-sulfate Heparin lyaseGlcN—N-sulfate MPS IIIB GlcNAc hexosaminidase GlcNAc MPS IIIB GlcNAcDeacetylase acetate MPS IIIB GlcNAc Heparin lyase GlcNAc MPS IIICGlcNAc-6-O sulfate 6-O sulfatase Sulfate MPS IIIC GlcNAc-6-O sulfate 6-Osulfatase hexosaminidase GlcNAc MPS IIIC GlcNAc-6-O sulfate 6-Osulfatase Heparin lyase GlcNAc MPS IIIC GlcNAc-6-O sulfate Heparin lyaseGlcNAc-6-O sulfate MPS IIID GlcN hexosaminidase GlcN MPS IIID GlcNHeparin lyase GlcN MPS IIID GlcN N-acetyl hexosaminidase GlcNActransferase MPS IVA Gal-6-O sulfate 6-O sulfatase Sulfate and GalNAc-6-Osulfate MPS IVA Gal-6-O sulfate galactosidase Gal-6-O sulfate andGalNAc-6-O sulfate MPS IVA Gal-6-O sulfate N-acetyl GalNAc-6-O sulfateand GalNAc-6-O galactosidase sulfate MPS IVA Gal-6-O sulfatehexosaminidase GalNAc-6-O sulfate and GalNAc-6-O sulfate MPS IVA Gal-6-Osulfate 6-O sulfatase galactosidase Gal and GalNAc-6-O sulfate MPS IVAGal-6-O sulfate 6-O sulfatase N-acetyl GalNAc and GalNAc-6-Ogalactosidase sulfate MPS IVA Gal-6-O sulfate Any combination GalNAc-6-Oand GalNAc-6-O of Chondroitin sulfate (+/−4-O sulfate (+/−4-O- lyase Aand/or sulfate) sulfate B and/or C activities) MPS IVA Gal-6-O sulfate6-O sulfatase Any combination GalNAc (+−4- and GalNAc-6-O of ChondroitinO sulfate) sulfate (+/−4-O- lyase A and/or sulfate B and/or Cactivities) MPS IVB Gal Galactosidase Gal MPS VI GalNAc-4-O 4-Osulfatase Sulfate sulfate MPS VI GalNAc-4-O 4-O sulfatase hexosaminidaseGalNAc sulfate MPS VI GalNAc-4-O 4-O sulfatase Chondroitin GalNAcsulfate lyase MPS VI GalNAc-4-O Chondroitin GalNAc-4-O sulfate lyasesulfate MPS VII GlcA β-glucuronidase GlcA Alpha Mannosidosis MannoseManosidase Man Aspartylglucosaminuria GlcNAc hexosaminidase GlcNAc FabryGalactose galactosidase Gal Fucosidosis Fucose fucosidase FucGalactosialidosis Galactose and/or Galactosidase Gal and/or Sialic acidand/or sialidase Sialic acid Gaucher glucose glucosidase glucose GM1gangliosidosis Beta-Galactose Beta-Galactosidase galactose GM1gangliosidosis Beta-Galactose Beta-Galactosidase Hexosaminidase GalNAcGM2 activator GalNAc hexosaminidase GalNAc deficiency Sialidosis Sialicacid Sialidase Sialic acid Sialidosis Sialic acid Alpha 2,3 Sialic acidSialidase Sialidosis Sialic acid Alphas 2,6 Sialic acid SialidaseSialidosis Sialic acid Alphas 2,8 Sialic acid Sialidase Krabbe Galactosegalactosidase Galactose Metachromatic Sulfated 3-O sulfatase SulfateLeukodystrophy galactosylceramide Metachromatic Sulfated 3-O sulfatasegalactosidase Galactose Leukodystrophy galactosylceramide MucolipidosisII Broad range of Any listed Any glycans enzyme monosaccharide orsulfate Mucolipidosis III Broad range of Any listed Any glycans enzymemonosaccharide or sulfate Mucolipidosis IV Broad range of Any listed Anyglycans enzyme monosaccharide or sulfate Multiple Sulfatase Sulfatedglycans sulfatase sulfate Deficiency Multiple Sulfatase Sulfated glycanssulfatase Any glycosidase monosaccharide Deficiency Multiple SulfataseSulfated glycans Any glycosidase Sulfated Deficiency monosaccharideGlycogen Storage glucose glucosidase glucose Disease (Pompe) SandhoffGalNAc hexosaminidase GalNAc Tay-Sachs GalNAc hexosaminidase GalNAc ABVariant GalNAc hexosaminidase GalNAc Schindler Disease Alpha-GalNAchexosaminidase GalNAc Salla Disease Sialic acid none Sialic Acid AlphaMannosidosis Alpha mannose mannosidase Mannose Beta Mannosidosis Betamannose mannosidase Mannose Globoid cell galactose galactosidasegalactose leukodystrophy

Provided herein are methods of diagnosing individuals (including, e.g.,a disease state or the severity of a disease states) with a cancerousdisease state or methods of monitoring the treatment of a cancer.Provided in Table 13 are specific embodiments of disease that areoptionally diagnosed and/or monitored according to various embodimentsdescribed herein. Table 13 also illustrates various non-limitingembodiments of specific enzyme(s) that are optionally utilized to treata biological sample from an individual suffering from or suspected of(e.g., through a pre- or preliminary screening process) suffering from acancerous disease state. Moreover, Table 13 further illustrates variousglycan residual compounds that are liberated in various embodimentsdescribed herein, such liberated glycan residual compounds optionallybeing detected and/or measured in order to diagnose and/or monitor acancerous disease state.

TABLE 13 Exemplary Oncology Uses Primary Secondary Glycan Non-ReducingLiberating Liberating Residual Cancer Type End Structure Enzyme EnzymeCompound Melanoma Sialic Acid Sialidase Sialic acid Melanoma Sialic AcidAlpha 2,8 Sialidase Sialic acid Melanoma Sialic Acid Alpha 2,3 SialidaseSialic acid Melanoma Sialic Acid Alpha 2,6 Sialidase Sialic acidMelanoma GalNAc Hexosaminidase GalNAc Melanoma GalNAc SialidaseHexosaminidase GalNAc Melanoma Sialic acid Hexosaminidase SialidaseSialic acid Melanoma Galactose galactosidase Galactose MelanomaGalactose sialidase galactosidase Galactose Melanoma Fucose fucosidaseFucose Melanoma Galactose Galactosidase Galactose Melanoma GlcNAchexosaminidase GlcNAc Melanoma Sulfate Sulfatase Sulfate MelanomaSulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAc MelanomaSulfated Sulfatase Iduronidase or IdoA or uronic acid glucouronidaseGlcA Neuroblastoma Sialic Acid Sialidase Sialic acid NeuroblastomaSialic Acid Alpha 2,8 Sialidase Sialic acid Neuroblastoma Sialic AcidAlpha 2,3 Sialidase Sialic acid Neuroblastoma Sialic Acid Alpha 2,6Sialidase Sialic acid Neuroblastoma GalNAc Hexosaminidase GalNAcNeuroblastoma GalNAc Sialidase Hexosaminidase GalNAc NeuroblastomaSialic acid Hexosaminidase Sialidase Sialic acid Neuroblastoma Galactosegalactosidase Galactose Neuroblastoma Galactose sialidase galactosidaseGalactose Neuroblastoma Fucose fucosidase Fucose Neuroblastoma GalactoseGalactosidase Galactose Neuroblastoma GlcNAc hexosaminidase GlcNAcNeuroblastoma Sulfate Sulfatase Sulfate Neuroblastoma Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Neuroblastoma Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA AdenocarcinomaSialic Acid Sialidase Sialic acid Adenocarcinoma Sialic Acid Alpha 2,8Sialidase Sialic acid Adenocarcinoma Sialic Acid Alpha 2,3 SialidaseSialic acid Adenocarcinoma Sialic Acid Alpha 2,6 Sialidase Sialic acidAdenocarcinoma GalNAc Hexosaminidase GalNAc Adenocarcinoma GalNAcSialidase Hexosaminidase GalNAc Adenocarcinoma Sialic acidHexosaminidase Sialidase Sialic acid Adenocarcinoma Galactosegalactosidase Galactose Adenocarcinoma Galactose sialidase galactosidaseGalactose Adenocarcinoma Fucose fucosidase Fucose AdenocarcinomaGalactose Galactosidase Galactose Adenocarcinoma GlcNAc hexosaminidaseGlcNAc Adenocarcinoma Sulfate Sulfatase Sulfate Adenocarcinoma SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Adenocarcinoma SulfatedSulfatase Iduronidase or IdoA or uronic acid glucouronidase GlcA MyelomaSialic Acid Sialidase Sialic acid Myeloma Sialic Acid Alpha 2,8Sialidase Sialic acid Myeloma Sialic Acid Alpha 2,3 Sialidase Sialicacid Myeloma Sialic Acid Alpha 2,6 Sialidase Sialic acid Myeloma GalNAcHexosaminidase GalNAc Myeloma GalNAc Sialidase Hexosaminidase GalNAcMyeloma Sialic acid Hexosaminidase Sialidase Sialic acid MyelomaGalactose galactosidase Galactose Myeloma Galactose sialidasegalactosidase Galactose Myeloma Fucose fucosidase Fucose MyelomaGalactose Galactosidase Galactose Myeloma GlcNAc hexosaminidase GlcNAcMyeloma Sulfate Sulfatase Sulfate Myeloma Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Myeloma Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA Breast SialicAcid Sialidase Sialic acid Breast Sialic Acid Alpha 2,8 Sialidase Sialicacid Breast Sialic Acid Alpha 2,3 Sialidase Sialic acid Breast SialicAcid Alpha 2,6 Sialidase Sialic acid Breast GalNAc Hexosaminidase GalNAcBreast GalNAc Sialidase Hexosaminidase GalNAc Breast Sialic acidHexosaminidase Sialidase Sialic acid Breast Galactose galactosidaseGalactose Breast Galactose sialidase galactosidase Galactose BreastFucose fucosidase Fucose Breast Galactose Galactosidase Galactose BreastGlcNAc hexosaminidase GlcNAc Breast Sulfate Sulfatase Sulfate BreastSulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAc BreastSulfated Sulfatase Iduronidase or IdoA or uronic acid glucouronidaseGlcA Ovarian Sialic Acid Sialidase Sialic acid Ovarian Sialic Acid Alpha2,8 Sialidase Sialic acid Ovarian Sialic Acid Alpha 2,3 Sialidase Sialicacid Ovarian Sialic Acid Alpha 2,6 Sialidase Sialic acid Ovarian GalNAcHexosaminidase GalNAc Ovarian GalNAc Sialidase Hexosaminidase GalNAcOvarian Sialic acid Hexosaminidase Sialidase Sialic acid OvarianGalactose galactosidase Galactose Ovarian Galactose sialidasegalactosidase Galactose Ovarian Fucose fucosidase Fucose OvarianGalactose Galactosidase Galactose Ovarian GlcNAc hexosaminidase GlcNAcOvarian Sulfate Sulfatase Sulfate Ovarian Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Ovarian Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA Stomach SialicAcid Sialidase Sialic acid Stomach Sialic Acid Alpha 2,8 SialidaseSialic acid Stomach Sialic Acid Alpha 2,3 Sialidase Sialic acid StomachSialic Acid Alpha 2,6 Sialidase Sialic acid Stomach GalNAcHexosaminidase GalNAc Stomach GalNAc Sialidase Hexosaminidase GalNAcStomach Sialic acid Hexosaminidase Sialidase Sialic acid StomachGalactose galactosidase Galactose Stomach Galactose sialidasegalactosidase Galactose Stomach Fucose fucosidase Fucose StomachGalactose Galactosidase Galactose Stomach GlcNAc hexosaminidase GlcNAcStomach Sulfate Sulfatase Sulfate Stomach Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Stomach Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA Lung Sialic AcidSialidase Sialic acid Lung Sialic Acid Alpha 2,8 Sialidase Sialic acidLung Sialic Acid Alpha 2,3 Sialidase Sialic acid Lung Sialic Acid Alpha2,6 Sialidase Sialic acid Lung GalNAc Hexosaminidase GalNAc Lung GalNAcSialidase Hexosaminidase GalNAc Lung Sialic acid HexosaminidaseSialidase Sialic acid Lung Galactose galactosidase Galactose LungGalactose sialidase galactosidase Galactose Lung Fucose fucosidaseFucose Lung Galactose Galactosidase Galactose Lung GlcNAc hexosaminidaseGlcNAc Lung Sulfate Sulfatase Sulfate Lung Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Lung Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA Pancreatic SialicAcid Sialidase Sialic acid Pancreatic Sialic Acid Alpha 2,8 SialidaseSialic acid Pancreatic Sialic Acid Alpha 2,3 Sialidase Sialic acidPancreatic Sialic Acid Alpha 2,6 Sialidase Sialic acid Pancreatic GalNAcHexosaminidase GalNAc Pancreatic GalNAc Sialidase Hexosaminidase GalNAcPancreatic Sialic acid Hexosaminidase Sialidase Sialic acid PancreaticGalactose galactosidase Galactose Pancreatic Galactose sialidasegalactosidase Galactose Pancreatic Fucose fucosidase Fucose PancreaticGalactose Galactosidase Galactose Pancreatic GlcNAc hexosaminidaseGlcNAc Pancreatic Sulfate Sulfatase Sulfate Pancreatic SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Pancreatic SulfatedSulfatase Iduronidase or IdoA or uronic acid glucouronidase GlcA OralSialic Acid Sialidase Sialic acid Oral Sialic Acid Alpha 2,8 SialidaseSialic acid Oral Sialic Acid Alpha 2,3 Sialidase Sialic acid Oral SialicAcid Alpha 2,6 Sialidase Sialic acid Oral GalNAc Hexosaminidase GalNAcOral GalNAc Sialidase Hexosaminidase GalNAc Oral Sialic acidHexosaminidase Sialidase Sialic acid Oral Galactose galactosidaseGalactose Oral Galactose sialidase galactosidase Galactose Oral Fucosefucosidase Fucose Oral Galactose Galactosidase Galactose Oral GlcNAchexosaminidase GlcNAc Oral Sulfate Sulfatase Sulfate Oral SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Oral Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA Colorectal SialicAcid Sialidase Sialic acid Colorectal Sialic Acid Alpha 2,8 SialidaseSialic acid Colorectal Sialic Acid Alpha 2,3 Sialidase Sialic acidColorectal Sialic Acid Alpha 2,6 Sialidase Sialic acid Colorectal GalNAcHexosaminidase GalNAc Colorectal GalNAc Sialidase Hexosaminidase GalNAcColorectal Sialic acid Hexosaminidase Sialidase Sialic acid ColorectalGalactose galactosidase Galactose Colorectal Galactose sialidasegalactosidase Galactose Colorectal Fucose fucosidase Fucose ColorectalGalactose Galactosidase Galactose Colorectal GlcNAc hexosaminidaseGlcNAc Colorectal Sulfate Sulfatase Sulfate Colorectal SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Colorectal SulfatedSulfatase Iduronidase or IdoA or uronic acid glucouronidase GlcA KidneySialic Acid Sialidase Sialic acid Kidney Sialic Acid Alpha 2,8 SialidaseSialic acid Kidney Sialic Acid Alpha 2,3 Sialidase Sialic acid KidneySialic Acid Alpha 2,6 Sialidase Sialic acid Kidney GalNAc HexosaminidaseGalNAc Kidney GalNAc Sialidase Hexosaminidase GalNAc Kidney Sialic acidHexosaminidase Sialidase Sialic acid Kidney Galactose galactosidaseGalactose Kidney Galactose sialidase galactosidase Galactose KidneyFucose fucosidase Fucose Kidney Galactose Galactosidase Galactose KidneyGlcNAc hexosaminidase GlcNAc Kidney Sulfate Sulfatase Sulfate KidneySulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAc KidneySulfated Sulfatase Iduronidase or IdoA or uronic acid glucouronidaseGlcA Bladder Sialic Acid Sialidase Sialic acid Bladder Sialic Acid Alpha2,8 Sialidase Sialic acid Bladder Sialic Acid Alpha 2,3 Sialidase Sialicacid Bladder Sialic Acid Alpha 2,6 Sialidase Sialic acid Bladder GalNAcHexosaminidase GalNAc Bladder GalNAc Sialidase Hexosaminidase GalNAcBladder Sialic acid Hexosaminidase Sialidase Sialic acid BladderGalactose galactosidase Galactose Bladder Galactose sialidasegalactosidase Galactose Bladder Fucose fucosidase Fucose BladderGalactose Galactosidase Galactose Bladder GlcNAc hexosaminidase GlcNAcBladder Sulfate Sulfatase Sulfate Bladder Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Bladder Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA Prostate SialicAcid Sialidase Sialic acid Prostate Sialic Acid Alpha 2,8 SialidaseSialic acid Prostate Sialic Acid Alpha 2,3 Sialidase Sialic acidProstate Sialic Acid Alpha 2,6 Sialidase Sialic acid Prostate GalNAcHexosaminidase GalNAc Prostate GalNAc Sialidase Hexosaminidase GalNAcProstate Sialic acid Hexosaminidase Sialidase Sialic acid ProstateGalactose galactosidase Galactose Prostate Galactose sialidasegalactosidase Galactose Prostate Fucose fucosidase Fucose ProstateGalactose Galactosidase Galactose Prostate GlcNAc hexosaminidase GlcNAcProstate Sulfate Sulfatase Sulfate Prostate Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Prostate Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA Uterine SialicAcid Sialidase Sialic acid Uterine Sialic Acid Alpha 2,8 SialidaseSialic acid Uterine Sialic Acid Alpha 2,3 Sialidase Sialic acid UterineSialic Acid Alpha 2,6 Sialidase Sialic acid Uterine GalNAcHexosaminidase GalNAc Uterine GalNAc Sialidase Hexosaminidase GalNAcUterine Sialic acid Hexosaminidase Sialidase Sialic acid UterineGalactose galactosidase Galactose Uterine Galactose sialidasegalactosidase Galactose Uterine Fucose fucosidase Fucose UterineGalactose Galactosidase Galactose Uterine GlcNAc hexosaminidase GlcNAcUterine Sulfate Sulfatase Sulfate Uterine Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Uterine Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA Thyroid SialicAcid Sialidase Sialic acid Thyroid Sialic Acid Alpha 2,8 SialidaseSialic acid Thyroid Sialic Acid Alpha 2,3 Sialidase Sialic acid ThyroidSialic Acid Alpha 2,6 Sialidase Sialic acid Thyroid GalNAcHexosaminidase GalNAc Thyroid GalNAc Sialidase Hexosaminidase GalNAcThyroid Sialic acid Hexosaminidase Sialidase Sialic acid ThyroidGalactose galactosidase Galactose Thyroid Galactose sialidasegalactosidase Galactose Thyroid Fucose fucosidase Fucose ThyroidGalactose Galactosidase Galactose Thyroid GlcNAc hexosaminidase GlcNAcThyroid Sulfate Sulfatase Sulfate Thyroid Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Thyroid Sulfated SulfataseIduronidase or IdoA or uronic acid glucouronidase GlcA Liver Sialic AcidSialidase Sialic acid Liver Sialic Acid Alpha 2,8 Sialidase Sialic acidLiver Sialic Acid Alpha 2,3 Sialidase Sialic acid Liver Sialic AcidAlpha 2,6 Sialidase Sialic acid Liver GalNAc Hexosaminidase GalNAc LiverGalNAc Sialidase Hexosaminidase GalNAc Liver Sialic acid HexosaminidaseSialidase Sialic acid Liver Galactose galactosidase Galactose LiverGalactose sialidase galactosidase Galactose Liver Fucose fucosidaseFucose Liver Galactose Galactosidase Galactose Liver GlcNAchexosaminidase GlcNAc Liver Sulfate Sulfatase Sulfate Liver SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Liver SulfatedSulfatase Iduronidase or IdoA or uronic acid glucouronidase GlcAEsophagus Sialic Acid Sialidase Sialic acid Esophagus Sialic Acid Alpha2,8 Sialidase Sialic acid Esophagus Sialic Acid Alpha 2,3 SialidaseSialic acid Esophagus Sialic Acid Alpha 2,6 Sialidase Sialic acidEsophagus GalNAc Hexosaminidase GalNAc Esophagus GalNAc SialidaseHexosaminidase GalNAc Esophagus Sialic acid Hexosaminidase SialidaseSialic acid Esophagus Galactose galactosidase Galactose EsophagusGalactose sialidase galactosidase Galactose Esophagus Fucose fucosidaseFucose Esophagus Galactose Galactosidase Galactose Esophagus GlcNAchexosaminidase GlcNAc Esophagus Sulfate Sulfatase Sulfate EsophagusSulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAc EsophagusSulfated Sulfatase Iduronidase or IdoA or uronic acid glucouronidaseGlcA Brain Sialic Acid Sialidase Sialic acid Brain Sialic Acid Alpha 2,8Sialidase Sialic acid Brain Sialic Acid Alpha 2,3 Sialidase Sialic acidBrain Sialic Acid Alpha 2,6 Sialidase Sialic acid Brain GalNAcHexosaminidase GalNAc Brain GalNAc Sialidase Hexosaminidase GalNAc BrainSialic acid Hexosaminidase Sialidase Sialic acid Brain Galactosegalactosidase Galactose Brain Galactose sialidase galactosidaseGalactose Brain Fucose fucosidase Fucose Brain Galactose GalactosidaseGalactose Brain GlcNAc hexosaminidase GlcNAc Brain Sulfate SulfataseSulfate Brain Sulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAcBrain Sulfated Sulfatase Iduronidase or IdoA or uronic acidglucouronidase GlcA Lymphomas Sialic Acid Sialidase Sialic acidLymphomas Sialic Acid Alpha 2,8 Sialidase Sialic acid Lymphomas SialicAcid Alpha 2,3 Sialidase Sialic acid Lymphomas Sialic Acid Alpha 2,6Sialidase Sialic acid Lymphomas GalNAc Hexosaminidase GalNAc LymphomasGalNAc Sialidase Hexosaminidase GalNAc Lymphomas Sialic acidHexosaminidase Sialidase Sialic acid Lymphomas Galactose galactosidaseGalactose Lymphomas Galactose sialidase galactosidase GalactoseLymphomas Fucose fucosidase Fucose Lymphomas Galactose GalactosidaseGalactose Lymphomas GlcNAc hexosaminidase GlcNAc Lymphomas SulfateSulfatase Sulfate Lymphomas Sulfated Sulfatase hexosaminidase GlcNAc orhexose GalNAc Lymphomas Sulfated Sulfatase Iduronidase or IdoA or uronicacid glucouronidase GlcA Leukemias Sialic Acid Sialidase Sialic acidLeukemias Sialic Acid Alpha 2,8 Sialidase Sialic acid Leukemias SialicAcid Alpha 2,3 Sialidase Sialic acid Leukemias Sialic Acid Alpha 2,6Sialidase Sialic acid Leukemias GalNAc Hexosaminidase GalNAc LeukemiasGalNAc Sialidase Hexosaminidase GalNAc Leukemias Sialic acidHexosaminidase Sialidase Sialic acid Leukemias Galactose galactosidaseGalactose Leukemias Galactose sialidase galactosidase GalactoseLeukemias Fucose fucosidase Fucose Leukemias Galactose GalactosidaseGalactose Leukemias GlcNAc hexosaminidase GlcNAc Leukemias SulfateSulfatase Sulfate Leukemias Sulfated Sulfatase hexosaminidase GlcNAc orhexose GalNAc Leukemias Sulfated Sulfatase Iduronidase or IdoA or uronicacid glucouronidase GlcA

Provided herein are methods of diagnosing individuals (including, e.g.,a disease state or the severity of a disease states) with a diseasestate associated with abnormal glycan accumulation. Provided in Table 14are specific embodiments of disease that are optionally diagnosed and/ormonitored according to various embodiments described herein. Table 14also illustrates various non-limiting embodiments of specific enzyme(s)that are optionally utilized to treat a biological sample from anindividual suffering from or suspected of (e.g., through a pre- orpreliminary screening process) suffering from various disease statesassociated with abnormal glycan accumulation. Moreover, Table 14 furtherillustrates various glycan residual compounds that are liberated invarious embodiments described herein, such liberated glycan residualcompounds optionally being detected and/or measured in order to diagnoseand/or monitor various disease states.

TABLE 14 Primary Secondary Glycan Non-Reducing Liberating LiberatingResidual Disease End Structure Enzyme Enzyme Compound Alzheimers SialicAcid Sialidase Sialic acid Alzheimers Sialic Acid Alpha 2,8 SialidaseSialic acid Alzheimers Sialic Acid Alpha 2,3 Sialidase Sialic acidAlzheimers Sialic Acid Alpha 2,6 Sialidase Sialic acid Alzheimers GalNAcHexosaminidase GalNAc Alzheimers GalNAc Sialidase Hexosaminidase GalNAcAlzheimers Sialic acid Hexosaminidase Sialidase Sialic acid AlzheimersGalactose galactosidase Galactose Alzheimers Galactose sialidasegalactosidase Galactose Alzheimers Fucose fucosidase Fucose AlzheimersGalactose Galactosidase Galactose Alzheimers GlcNAc hexosaminidaseGlcNAc Alzheimers Sulfate Sulfatase Sulfate Alzheimers SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Alzheimers SulfatedSulfatase Iduronidase or IdoA or GlcA uronic acid glucuronidaseAmyotrophic Lateral Sialic Acid Sialidase Sialic acid SclerosisAmyotrophic Lateral Sialic Acid Alpha 2,8 Sialidase Sialic acidSclerosis Amyotrophic Lateral Sialic Acid Alpha 2,3 Sialidase Sialicacid Sclerosis Amyotrophic Lateral Sialic Acid Alpha 2,6 SialidaseSialic acid Sclerosis Amyotrophic Lateral GalNAc Hexosaminidase GalNAcSclerosis Amyotrophic Lateral GalNAc Sialidase Hexosaminidase GalNAcSclerosis Amyotrophic Lateral Sialic acid Hexosaminidase SialidaseSialic acid Sclerosis Amyotrophic Lateral Galactose galactosidaseGalactose Sclerosis Amyotrophic Lateral Galactose sialidasegalactosidase Galactose Sclerosis Amyotrophic Lateral Fucose fucosidaseFucose Sclerosis Amyotrophic Lateral Galactose Galactosidase GalactoseSclerosis Amyotrophic Lateral GlcNAc hexosaminidase GlcNAc SclerosisAmyotrophic Lateral Sulfate Sulfatase Sulfate Sclerosis AmyotrophicLateral Sulfated Sulfatase hexosaminidase GlcNAc or Sclerosis hexoseGalNAc Amyotrophic Lateral Sulfated Sulfatase Iduronidase or IdoA orGlcA Sclerosis uronic acid glucuronidase Cerebral Palsy Sialic AcidSialidase Sialic acid Cerebral Palsy Sialic Acid Alpha 2,8 SialidaseSialic acid Cerebral Palsy Sialic Acid Alpha 2,3 Sialidase Sialic acidCerebral Palsy Sialic Acid Alpha 2,6 Sialidase Sialic acid CerebralPalsy GalNAc Hexosaminidase GalNAc Cerebral Palsy GalNAc SialidaseHexosaminidase GalNAc Cerebral Palsy Sialic acid HexosaminidaseSialidase Sialic acid Cerebral Palsy Galactose galactosidase GalactoseCerebral Palsy Galactose sialidase galactosidase Galactose CerebralPalsy Fucose fucosidase Fucose Cerebral Palsy Galactose GalactosidaseGalactose Cerebral Palsy GlcNAc hexosaminidase GlcNAc Cerebral PalsySulfate Sulfatase Sulfate Cerebral Palsy Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Cerebral Palsy Sulfated SulfataseIduronidase or IdoA or GlcA uronic acid glucuronidase SchizophreniaSialic Acid Sialidase Sialic acid Schizophrenia Sialic Acid Alpha 2,8Sialidase Sialic acid Schizophrenia Sialic Acid Alpha 2,3 SialidaseSialic acid Schizophrenia Sialic Acid Alpha 2,6 Sialidase Sialic acidSchizophrenia GalNAc Hexosaminidase GalNAc Schizophrenia GalNAcSialidase Hexosaminidase GalNAc Schizophrenia Sialic acid HexosaminidaseSialidase Sialic acid Schizophrenia Galactose galactosidase GalactoseSchizophrenia Galactose sialidase galactosidase Galactose SchizophreniaFucose fucosidase Fucose Schizophrenia Galactose Galactosidase GalactoseSchizophrenia GlcNAc hexosaminidase GlcNAc Schizophrenia SulfateSulfatase Sulfate Schizophrenia Sulfated Sulfatase hexosaminidase GlcNAcor hexose GalNAc Schizophrenia Sulfated Sulfatase Iduronidase or IdoA orGlcA uronic acid glucouronidase Bipolar Disorder Sialic Acid SialidaseSialic acid Bipolar Disorder Sialic Acid Alpha 2,8 Sialidase Sialic acidBipolar Disorder Sialic Acid Alpha 2,3 Sialidase Sialic acid BipolarDisorder Sialic Acid Alpha 2,6 Sialidase Sialic acid Bipolar DisorderGalNAc Hexosaminidase GalNAc Bipolar Disorder GalNAc SialidaseHexosaminidase GalNAc Bipolar Disorder Sialic acid HexosaminidaseSialidase Sialic acid Bipolar Disorder Galactose galactosidase GalactoseBipolar Disorder Galactose sialidase galactosidase Galactose BipolarDisorder Fucose fucosidase Fucose Bipolar Disorder GalactoseGalactosidase Galactose Bipolar Disorder GlcNAc hexosaminidase GlcNAcBipolar Disorder Sulfate Sulfatase Sulfate Bipolar Disorder SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Bipolar DisorderSulfated Sulfatase Iduronidase or IdoA or GlcA uronic acidglucouronidase Depression Sialic Acid Sialidase Sialic acid DepressionSialic Acid Alpha 2,8 Sialidase Sialic acid Depression Sialic Acid Alpha2,3 Sialidase Sialic acid Depression Sialic Acid Alpha 2,6 SialidaseSialic acid Depression GalNAc Hexosaminidase GalNAc Depression GalNAcSialidase Hexosaminidase GalNAc Depression Sialic acid HexosaminidaseSialidase Sialic acid Depression Galactose galactosidase GalactoseDepression Galactose sialidase galactosidase Galactose Depression Fucosefucosidase Fucose Depression Galactose Galactosidase GalactoseDepression GlcNAc hexosaminidase GlcNAc Depression Sulfate SulfataseSulfate Depression Sulfated Sulfatase hexosaminidase GlcNAc or hexoseGalNAc Depression Sulfated Sulfatase Iduronidase or IdoA or GlcA uronicacid glucouronidase Epilepsy Sialic Acid Sialidase Sialic acid EpilepsySialic Acid Alpha 2,8 Sialidase Sialic acid Epilepsy Sialic Acid Alpha2,3 Sialidase Sialic acid Epilepsy Sialic Acid Alpha 2,6 SialidaseSialic acid Epilepsy GalNAc Hexosaminidase GalNAc Epilepsy GalNAcSialidase Hexosaminidase GalNAc Epilepsy Sialic acid HexosaminidaseSialidase Sialic acid Epilepsy Galactose galactosidase GalactoseEpilepsy Galactose sialidase galactosidase Galactose Epilepsy Fucosefucosidase Fucose Epilepsy Galactose Galactosidase Galactose EpilepsyGlcNAc hexosaminidase GlcNAc Epilepsy Sulfate Sulfatase Sulfate EpilepsySulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAc EpilepsySulfated Sulfatase Iduronidase or IdoA or GlcA uronic acidglucouronidase Migraine Sialic Acid Sialidase Sialic acid MigraineSialic Acid Alpha 2,8 Sialidase Sialic acid Migraine Sialic Acid Alpha2,3 Sialidase Sialic acid Migraine Sialic Acid Alpha 2,6 SialidaseSialic acid Migraine GalNAc Hexosaminidase GalNAc Migraine GalNAcSialidase Hexosaminidase GalNAc Migraine Sialic acid HexosaminidaseSialidase Sialic acid Migraine Galactose galactosidase GalactoseMigraine Galactose sialidase galactosidase Galactose Migraine Fucosefucosidase Fucose Migraine Galactose Galactosidase Galactose MigraineGlcNAc hexosaminidase GlcNAc Migraine Sulfate Sulfatase Sulfate MigraineSulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAc MigraineSulfated Sulfatase Iduronidase or IdoA or GlcA uronic acidglucouronidase Multiple Sclerosis Sialic Acid Sialidase Sialic acidMultiple Sclerosis Sialic Acid Alpha 2,8 Sialidase Sialic acid MultipleSclerosis Sialic Acid Alpha 2,3 Sialidase Sialic acid Multiple SclerosisSialic Acid Alpha 2,6 Sialidase Sialic acid Multiple Sclerosis GalNAcHexosaminidase GalNAc Multiple Sclerosis GalNAc Sialidase HexosaminidaseGalNAc Multiple Sclerosis Sialic acid Hexosaminidase Sialidase Sialicacid Multiple Sclerosis Galactose galactosidase Galactose MultipleSclerosis Galactose sialidase galactosidase Galactose Multiple SclerosisFucose fucosidase Fucose Multiple Sclerosis Galactose GalactosidaseGalactose Multiple Sclerosis GlcNAc hexosaminidase GlcNAc MultipleSclerosis Sulfate Sulfatase Sulfate Multiple Sclerosis SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Multiple SclerosisSulfated Sulfatase Iduronidase or IdoA or GlcA uronic acidglucouronidase Parkinson's Sialic Acid Sialidase Sialic acid Parkinson'sSialic Acid Alpha 2,8 Sialidase Sialic acid Parkinson's Sialic AcidAlpha 2,3 Sialidase Sialic acid Parkinson's Sialic Acid Alpha 2,6Sialidase Sialic acid Parkinson's GalNAc Hexosaminidase GalNAcParkinson's GalNAc Sialidase Hexosaminidase GalNAc Parkinson's Sialicacid Hexosaminidase Sialidase Sialic acid Parkinson's Galactosegalactosidase Galactose Parkinson's Galactose sialidase galactosidaseGalactose Parkinson's Fucose fucosidase Fucose Parkinson's GalactoseGalactosidase Galactose Parkinson's GlcNAc hexosaminidase GlcNAcParkinson's Sulfate Sulfatase Sulfate Parkinson's Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Parkinson's Sulfated SulfataseIduronidase or IdoA or GlcA uronic acid glucouronidase RheumatoidArthritis Sialic Acid Sialidase Sialic acid Rheumatoid Arthritis SialicAcid Alpha 2,8 Sialidase Sialic acid Rheumatoid Arthritis Sialic AcidAlpha 2,3 Sialidase Sialic acid Rheumatoid Arthritis Sialic Acid Alpha2,6 Sialidase Sialic acid Rheumatoid Arthritis GalNAc HexosaminidaseGalNAc Rheumatoid Arthritis GalNAc Sialidase Hexosaminidase GalNAcRheumatoid Arthritis Sialic acid Hexosaminidase Sialidase Sialic acidRheumatoid Arthritis Galactose galactosidase Galactose RheumatoidArthritis Galactose sialidase galactosidase Galactose RheumatoidArthritis Fucose fucosidase Fucose Rheumatoid Arthritis GalactoseGalactosidase Galactose Rheumatoid Arthritis GlcNAc hexosaminidaseGlcNAc Rheumatoid Arthritis Sulfate Sulfatase Sulfate RheumatoidArthritis Sulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAcRheumatoid Arthritis Sulfated Sulfatase Iduronidase or IdoA or GlcAuronic acid glucouronidase Psoriatic Arthritis Sialic Acid SialidaseSialic acid Psoriatic Arthritis Sialic Acid Alpha 2,8 Sialidase Sialicacid Psoriatic Arthritis Sialic Acid Alpha 2,3 Sialidase Sialic acidPsoriatic Arthritis Sialic Acid Alpha 2,6 Sialidase Sialic acidPsoriatic Arthritis GalNAc Hexosaminidase GalNAc Psoriatic ArthritisGalNAc Sialidase Hexosaminidase GalNAc Psoriatic Arthritis Sialic acidHexosaminidase Sialidase Sialic acid Psoriatic Arthritis Galactosegalactosidase Galactose Psoriatic Arthritis Galactose sialidasegalactosidase Galactose Psoriatic Arthritis Fucose fucosidase FucosePsoriatic Arthritis Galactose Galactosidase Galactose PsoriaticArthritis GlcNAc hexosaminidase GlcNAc Psoriatic Arthritis SulfateSulfatase Sulfate Psoriatic Arthritis Sulfated Sulfatase hexosaminidaseGlcNAc or hexose GalNAc Psoriatic Arthritis Sulfated SulfataseIduronidase or IdoA or GlcA uronic acid glucouronidase Asthma SialicAcid Sialidase Sialic acid Asthma Sialic Acid Alpha 2,8 Sialidase Sialicacid Asthma Sialic Acid Alpha 2,3 Sialidase Sialic acid Asthma SialicAcid Alpha 2,6 Sialidase Sialic acid Asthma GalNAc Hexosaminidase GalNAcAsthma GalNAc Sialidase Hexosaminidase GalNAc Asthma Sialic acidHexosaminidase Sialidase Sialic acid Asthma Galactose galactosidaseGalactose Asthma Galactose sialidase galactosidase Galactose AsthmaFucose fucosidase Fucose Asthma Galactose Galactosidase Galactose AsthmaGlcNAc hexosaminidase GlcNAc Asthma Sulfate Sulfatase Sulfate AsthmaSulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAc AsthmaSulfated Sulfatase Iduronidase or IdoA or GlcA uronic acidglucouronidase Chronic Obstructive Sialic Acid Sialidase Sialic acidPulmonary Disorder Chronic Obstructive Sialic Acid Alpha 2,8 SialidaseSialic acid Pulmonary Disorder Chronic Obstructive Sialic Acid Alpha 2,3Sialidase Sialic acid Pulmonary Disorder Chronic Obstructive Sialic AcidAlpha 2,6 Sialidase Sialic acid Pulmonary Disorder Chronic ObstructiveGalNAc Hexosaminidase GalNAc Pulmonary Disorder Chronic ObstructiveGalNAc Sialidase Hexosaminidase GalNAc Pulmonary Disorder ChronicObstructive Sialic acid Hexosaminidase Sialidase Sialic acid PulmonaryDisorder Chronic Obstructive Galactose galactosidase Galactose PulmonaryDisorder Chronic Obstructive Galactose sialidase galactosidase GalactosePulmonary Disorder Chronic Obstructive Fucose fucosidase FucosePulmonary Disorder Chronic Obstructive Galactose Galactosidase GalactosePulmonary Disorder Chronic Obstructive GlcNAc hexosaminidase GlcNAcPulmonary Disorder Chronic Obstructive Sulfate Sulfatase SulfatePulmonary Disorder Chronic Obstructive Sulfated Sulfatase hexosaminidaseGlcNAc or Pulmonary Disorder hexose GalNAc Chronic Obstructive SulfatedSulfatase Iduronidase or IdoA or GlcA Pulmonary Disorder uronic acidglucouronidase Lupus Sialic Acid Sialidase Sialic acid Lupus Sialic AcidAlpha 2,8 Sialidase Sialic acid Lupus Sialic Acid Alpha 2,3 SialidaseSialic acid Lupus Sialic Acid Alpha 2,6 Sialidase Sialic acid LupusGalNAc Hexosaminidase GalNAc Lupus GalNAc Sialidase HexosaminidaseGalNAc Lupus Sialic acid Hexosaminidase Sialidase Sialic acid LupusGalactose galactosidase Galactose Lupus Galactose sialidasegalactosidase Galactose Lupus Fucose fucosidase Fucose Lupus GalactoseGalactosidase Galactose Lupus GlcNAc hexosaminidase GlcNAc Lupus SulfateSulfatase Sulfate Lupus Sulfated Sulfatase hexosaminidase GlcNAc orhexose GalNAc Lupus Sulfated Sulfatase Iduronidase or IdoA or GlcAuronic acid glucouronidase Hepatitis Sialic Acid Sialidase Sialic acidHepatitis Sialic Acid Alpha 2,8 Sialidase Sialic acid Hepatitis SialicAcid Alpha 2,3 Sialidase Sialic acid Hepatitis Sialic Acid Alpha 2,6Sialidase Sialic acid Hepatitis GalNAc Hexosaminidase GalNAc HepatitisGalNAc Sialidase Hexosaminidase GalNAc Hepatitis Sialic acidHexosaminidase Sialidase Sialic acid Hepatitis Galactose galactosidaseGalactose Hepatitis Galactose sialidase galactosidase GalactoseHepatitis Fucose fucosidase Fucose Hepatitis Galactose GalactosidaseGalactose Hepatitis GlcNAc hexosaminidase GlcNAc Hepatitis SulfateSulfatase Sulfate Hepatitis Sulfated Sulfatase hexosaminidase GlcNAc orhexose GalNAc Hepatitis Sulfated Sulfatase Iduronidase or IdoA or GlcAuronic acid glucouronidase Renal Disease Sialic Acid Sialidase Sialicacid Renal Disease Sialic Acid Alpha 2,8 Sialidase Sialic acid RenalDisease Sialic Acid Alpha 2,3 Sialidase Sialic acid Renal Disease SialicAcid Alpha 2,6 Sialidase Sialic acid Renal Disease GalNAc HexosaminidaseGalNAc Renal Disease GalNAc Sialidase Hexosaminidase GalNAc RenalDisease Sialic acid Hexosaminidase Sialidase Sialic acid Renal DiseaseGalactose galactosidase Galactose Renal Disease Galactose sialidasegalactosidase Galactose Renal Disease Fucose fucosidase Fucose RenalDisease Galactose Galactosidase Galactose Renal Disease GlcNAchexosaminidase GlcNAc Renal Disease Sulfate Sulfatase Sulfate RenalDisease Sulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAc RenalDisease Sulfated Sulfatase Iduronidase or IdoA or GlcA uronic acidglucouronidase Sickle Cell Disease Sialic Acid Sialidase Sialic acidSickle Cell Disease Sialic Acid Alpha 2,8 Sialidase Sialic acid SickleCell Disease Sialic Acid Alpha 2,3 Sialidase Sialic acid Sickle CellDisease Sialic Acid Alpha 2,6 Sialidase Sialic acid Sickle Cell DiseaseGalNAc Hexosaminidase GalNAc Sickle Cell Disease GalNAc SialidaseHexosaminidase GalNAc Sickle Cell Disease Sialic acid HexosaminidaseSialidase Sialic acid Sickle Cell Disease Galactose galactosidaseGalactose Sickle Cell Disease Galactose sialidase galactosidaseGalactose Sickle Cell Disease Fucose fucosidase Fucose Sickle CellDisease Galactose Galactosidase Galactose Sickle Cell Disease GlcNAchexosaminidase GlcNAc Sickle Cell Disease Sulfate Sulfatase SulfateSickle Cell Disease Sulfated Sulfatase hexosaminidase GlcNAc or hexoseGalNAc Sickle Cell Disease Sulfated Sulfatase Iduronidase or IdoA orGlcA uronic acid glucouronidase Fibromyalgia Sialic Acid SialidaseSialic acid Fibromyalgia Sialic Acid Alpha 2,8 Sialidase Sialic acidFibromyalgia Sialic Acid Alpha 2,3 Sialidase Sialic acid FibromyalgiaSialic Acid Alpha 2,6 Sialidase Sialic acid Fibromyalgia GalNAcHexosaminidase GalNAc Fibromyalgia GalNAc Sialidase HexosaminidaseGalNAc Fibromyalgia Sialic acid Hexosaminidase Sialidase Sialic acidFibromyalgia Galactose galactosidase Galactose Fibromyalgia Galactosesialidase galactosidase Galactose Fibromyalgia Fucose fucosidase FucoseFibromyalgia Galactose Galactosidase Galactose Fibromyalgia GlcNAchexosaminidase GlcNAc Fibromyalgia Sulfate Sulfatase SulfateFibromyalgia Sulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAcFibromyalgia Sulfated Sulfatase Iduronidase or IdoA or GlcA uronic acidglucouronidase Irritable Bowel Sialic Acid Sialidase Sialic acidSyndrome Irritable Bowel Sialic Acid Alpha 2,8 Sialidase Sialic acidSyndrome Irritable Bowel Sialic Acid Alpha 2,3 Sialidase Sialic acidSyndrome Irritable Bowel Sialic Acid Alpha 2,6 Sialidase Sialic acidSyndrome Irritable Bowel GalNAc Hexosaminidase GalNAc Syndrome IrritableBowel GalNAc Sialidase Hexosaminidase GalNAc Syndrome Irritable BowelSialic acid Hexosaminidase Sialidase Sialic acid Syndrome IrritableBowel Galactose galactosidase Galactose Syndrome Irritable BowelGalactose sialidase galactosidase Galactose Syndrome Irritable BowelFucose fucosidase Fucose Syndrome Irritable Bowel GalactoseGalactosidase Galactose Syndrome Irritable Bowel GlcNAc hexosaminidaseGlcNAc Syndrome Irritable Bowel Sulfate Sulfatase Sulfate SyndromeIrritable Bowel Sulfated Sulfatase hexosaminidase GlcNAc or Syndromehexose GalNAc Irritable Bowel Sulfated Sulfatase Iduronidase or IdoA orGlcA Syndrome uronic acid glucouronidase Ulcer Sialic Acid SialidaseSialic acid Ulcer Sialic Acid Alpha 2,8 Sialidase Sialic acid UlcerSialic Acid Alpha 2,3 Sialidase Sialic acid Ulcer Sialic Acid Alpha 2,6Sialidase Sialic acid Ulcer GalNAc Hexosaminidase GalNAc Ulcer GalNAcSialidase Hexosaminidase GalNAc Ulcer Sialic acid HexosaminidaseSialidase Sialic acid Ulcer Galactose galactosidase Galactose UlcerGalactose sialidase galactosidase Galactose Ulcer Fucose fucosidaseFucose Ulcer Galactose Galactosidase Galactose Ulcer GlcNAchexosaminidase GlcNAc Ulcer Sulfate Sulfatase Sulfate Ulcer SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Ulcer SulfatedSulfatase Iduronidase or IdoA or GlcA uronic acid glucouronidaseIrritable Bowel Disease Sialic Acid Sialidase Sialic acid IrritableBowel Disease Sialic Acid Alpha 2,8 Sialidase Sialic acid IrritableBowel Disease Sialic Acid Alpha 2,3 Sialidase Sialic acid IrritableBowel Disease Sialic Acid Alpha 2,6 Sialidase Sialic acid IrritableBowel Disease GalNAc Hexosaminidase GalNAc Irritable Bowel DiseaseGalNAc Sialidase Hexosaminidase GalNAc Irritable Bowel Disease Sialicacid Hexosaminidase Sialidase Sialic acid Irritable Bowel DiseaseGalactose galactosidase Galactose Irritable Bowel Disease Galactosesialidase galactosidase Galactose Irritable Bowel Disease Fucosefucosidase Fucose Irritable Bowel Disease Galactose GalactosidaseGalactose Irritable Bowel Disease GlcNAc hexosaminidase GlcNAc IrritableBowel Disease Sulfate Sulfatase Sulfate Irritable Bowel Disease SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Irritable Bowel DiseaseSulfated Sulfatase Iduronidase or IdoA or GlcA uronic acidglucouronidase Coronary Artery Disease Sialic Acid Sialidase Sialic acidCoronary Artery Disease Sialic Acid Alpha 2,8 Sialidase Sialic acidCoronary Artery Disease Sialic Acid Alpha 2,3 Sialidase Sialic acidCoronary Artery Disease Sialic Acid Alpha 2,6 Sialidase Sialic acidCoronary Artery Disease GalNAc Hexosaminidase GalNAc Coronary ArteryDisease GalNAc Sialidase Hexosaminidase GalNAc Coronary Artery DiseaseSialic acid Hexosaminidase Sialidase Sialic acid Coronary Artery DiseaseGalactose galactosidase Galactose Coronary Artery Disease Galactosesialidase galactosidase Galactose Coronary Artery Disease Fucosefucosidase Fucose Coronary Artery Disease Galactose GalactosidaseGalactose Coronary Artery Disease GlcNAc hexosaminidase GlcNAc CoronaryArtery Disease Sulfate Sulfatase Sulfate Coronary Artery DiseaseSulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAc CoronaryArtery Disease Sulfated Sulfatase Iduronidase or IdoA or GlcA uronicacid glucouronidase Restenosis Sialic Acid Sialidase Sialic acidRestenosis Sialic Acid Alpha 2,8 Sialidase Sialic acid Restenosis SialicAcid Alpha 2,3 Sialidase Sialic acid Restenosis Sialic Acid Alpha 2,6Sialidase Sialic acid Restenosis GalNAc Hexosaminidase GalNAc RestenosisGalNAc Sialidase Hexosaminidase GalNAc Restenosis Sialic acidHexosaminidase Sialidase Sialic acid Restenosis Galactose galactosidaseGalactose Restenosis Galactose sialidase galactosidase GalactoseRestenosis Fucose fucosidase Fucose Restenosis Galactose GalactosidaseGalactose Restenosis GlcNAc hexosaminidase GlcNAc Restenosis SulfateSulfatase Sulfate Restenosis Sulfated Sulfatase hexosaminidase GlcNAc orhexose GalNAc Restenosis Sulfated Sulfatase Iduronidase or IdoA or GlcAuronic acid glucouronidase Stroke Sialic Acid Sialidase Sialic acidStroke Sialic Acid Alpha 2,8 Sialidase Sialic acid Stroke Sialic AcidAlpha 2,3 Sialidase Sialic acid Stroke Sialic Acid Alpha 2,6 SialidaseSialic acid Stroke GalNAc Hexosaminidase GalNAc Stroke GalNAc SialidaseHexosaminidase GalNAc Stroke Sialic acid Hexosaminidase Sialidase Sialicacid Stroke Galactose galactosidase Galactose Stroke Galactose sialidasegalactosidase Galactose Stroke Fucose fucosidase Fucose Stroke GalactoseGalactosidase Galactose Stroke GlcNAc hexosaminidase GlcNAc StrokeSulfate Sulfatase Sulfate Stroke Sulfated Sulfatase hexosaminidaseGlcNAc or hexose GalNAc Stroke Sulfated Sulfatase Iduronidase or IdoA orGlcA uronic acid glucouronidase Diabetes Sialic Acid Sialidase Sialicacid Diabetes Sialic Acid Alpha 2,8 Sialidase Sialic acid DiabetesSialic Acid Alpha 2,3 Sialidase Sialic acid Diabetes Sialic Acid Alpha2,6 Sialidase Sialic acid Diabetes GalNAc Hexosaminidase GalNAc DiabetesGalNAc Sialidase Hexosaminidase GalNAc Diabetes Sialic acidHexosaminidase Sialidase Sialic acid Diabetes Galactose galactosidaseGalactose Diabetes Galactose sialidase galactosidase Galactose DiabetesFucose fucosidase Fucose Diabetes Galactose Galactosidase GalactoseDiabetes GlcNAc hexosaminidase GlcNAc Diabetes Sulfate Sulfatase SulfateDiabetes Sulfated Sulfatase hexosaminidase GlcNAc or hexose GalNAcDiabetes Sulfated Sulfatase Iduronidase or IdoA or GlcA uronic acidglucouronidase Hyperheparanemia Sialic Acid Sialidase Sialic acidHyperheparanemia Sialic Acid Alpha 2,8 Sialidase Sialic acidHyperheparanemia Sialic Acid Alpha 2,3 Sialidase Sialic acidHyperheparanemia Sialic Acid Alpha 2,6 Sialidase Sialic acidHyperheparanemia GalNAc Hexosaminidase GalNAc Hyperheparanemia GalNAcSialidase Hexosaminidase GalNAc Hyperheparanemia Sialic acidHexosaminidase Sialidase Sialic acid Hyperheparanemia Galactosegalactosidase Galactose Hyperheparanemia Galactose sialidasegalactosidase Galactose Hyperheparanemia Fucose fucosidase FucoseHyperheparanemia Galactose Galactosidase Galactose HyperheparanemiaGlcNAc hexosaminidase GlcNAc Hyperheparanemia Sulfate Sulfatase SulfateHyperheparanemia Sulfated Sulfatase hexosaminidase GlcNAc or hexoseGalNAc Hyperheparanemia Sulfated Sulfatase Iduronidase or IdoA or GlcAuronic acid glucouronidase Hypergangliosidemia Sialic Acid SialidaseSialic acid Hypergangliosidemia Sialic Acid Alpha 2,8 Sialidase Sialicacid Hypergangliosidemia Sialic Acid Alpha 2,3 Sialidase Sialic acidHypergangliosidemia Sialic Acid Alpha 2,6 Sialidase Sialic acidHypergangliosidemia GalNAc Hexosaminidase GalNAc HypergangliosidemiaGalNAc Sialidase Hexosaminidase GalNAc Hypergangliosidemia Sialic acidHexosaminidase Sialidase Sialic acid Hypergangliosidemia Galactosegalactosidase Galactose Hypergangliosidemia Galactose sialidasegalactosidase Galactose Hypergangliosidemia Fucose fucosidase FucoseHypergangliosidemia Galactose Galactosidase GalactoseHypergangliosidemia GlcNAc hexosaminidase GlcNAc HypergangliosidemiaSulfate Sulfatase Sulfate Hypergangliosidemia Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Hypergangliosidemia SulfatedSulfatase Iduronidase or IdoA or GlcA uronic acid glucouronidaseHypermucinemia Sialic Acid Sialidase Sialic acid Hypermucinemia SialicAcid Alpha 2,8 Sialidase Sialic acid Hypermucinemia Sialic Acid Alpha2,3 Sialidase Sialic acid Hypermucinemia Sialic Acid Alpha 2,6 SialidaseSialic acid Hypermucinemia GalNAc Hexosaminidase GalNAc HypermucinemiaGalNAc Sialidase Hexosaminidase GalNAc Hypermucinemia Sialic acidHexosaminidase Sialidase Sialic acid Hypermucinemia Galactosegalactosidase Galactose Hypermucinemia Galactose sialidase galactosidaseGalactose Hypermucinemia Fucose fucosidase Fucose HypermucinemiaGalactose Galactosidase Galactose Hypermucinemia GlcNAc hexosaminidaseGlcNAc Hypermucinemia Sulfate Sulfatase Sulfate Hypermucinemia SulfatedSulfatase hexosaminidase GlcNAc or hexose GalNAc Hypermucinemia SulfatedSulfatase Iduronidase or IdoA or GlcA uronic acid glucouronidase HyperO-linked Sialic Acid Sialidase Sialic acid glycanemia Hyper O-linkedSialic Acid Alpha 2,8 Sialidase Sialic acid glycanemia Hyper O-linkedSialic Acid Alpha 2,3 Sialidase Sialic acid glycanemia Hyper O-linkedSialic Acid Alpha 2,6 Sialidase Sialic acid glycanemia Hyper O-linkedGalNAc Hexosaminidase GalNAc glycanemia Hyper O-linked GalNAc SialidaseHexosaminidase GalNAc glycanemia Hyper O-linked Sialic acidHexosaminidase Sialidase Sialic acid glycanemia Hyper O-linked Galactosegalactosidase Galactose glycanemia Hyper O-linked Galactose sialidasegalactosidase Galactose glycanemia Hyper O-linked Fucose fucosidaseFucose glycanemia Hyper O-linked Galactose Galactosidase Galactoseglycanemia Hyper O-linked GlcNAc hexosaminidase GlcNAc glycanemia HyperO-linked Sulfate Sulfatase Sulfate glycanemia Hyper O-linked SulfatedSulfatase hexosaminidase GlcNAc or glycanemia hexose GalNAc HyperO-linked Sulfated Sulfatase Iduronidase or IdoA or GlcA glycanemiauronic acid glucouronidase Hyper N-linked Sialic Acid Sialidase Sialicacid glycanemia Hyper N-linked Sialic Acid Alpha 2,8 Sialidase Sialicacid glycanemia Hyper N-linked Sialic Acid Alpha 2,3 Sialidase Sialicacid glycanemia Hyper N-linked Sialic Acid Alpha 2,6 Sialidase Sialicacid glycanemia Hyper N-linked GalNAc Hexosaminidase GalNAc glycanemiaHyper N-linked GalNAc Sialidase Hexosaminidase GalNAc glycanemia HyperN-linked Sialic acid Hexosaminidase Sialidase Sialic acid glycanemiaHyper N-linked Galactose galactosidase Galactose glycanemia HyperN-linked Galactose sialidase galactosidase Galactose glycanemia HyperN-linked Fucose fucosidase Fucose glycanemia Hyper N-linked GalactoseGalactosidase Galactose glycanemia Hyper N-linked GlcNAc hexosaminidaseGlcNAc glycanemia Hyper N-linked Sulfate Sulfatase Sulfate glycanemiaHyper N-linked Sulfated Sulfatase hexosaminidase GlcNAc or glycanemiahexose GalNAc Hyper N-linked Sulfated Sulfatase Iduronidase or IdoA orGlcA glycanemia uronic acid glucouronidase Hypersialylemia Sialic AcidSialidase Sialic acid Hypersialylemia Sialic Acid Alpha 2,8 SialidaseSialic acid Hypersialylemia Sialic Acid Alpha 2,3 Sialidase Sialic acidHypersialylemia Sialic Acid Alpha 2,6 Sialidase Sialic acidHypersialylemia GalNAc Hexosaminidase GalNAc Hypersialylemia GalNAcSialidase Hexosaminidase GalNAc Hypersialylemia Sialic acidHexosaminidase Sialidase Sialic acid Hypersialylemia Galactosegalactosidase Galactose Hypersialylemia Galactose sialidasegalactosidase Galactose Hypersialylemia Fucose fucosidase FucoseHypersialylemia Galactose Galactosidase Galactose Hypersialylemia GlcNAchexosaminidase GlcNAc Hypersialylemia Sulfate Sulfatase SulfateHypersialylemia Sulfated Sulfatase hexosaminidase GlcNAc or hexoseGalNAc Hypersialylemia Sulfated Sulfatase Iduronidase or IdoA or GlcAuronic acid glucouronidase Hyperfucosylemia Sialic Acid Sialidase Sialicacid Hyperfucosylemia Sialic Acid Alpha 2,8 Sialidase Sialic acidHyperfucosylemia Sialic Acid Alpha 2,3 Sialidase Sialic acidHyperfucosylemia Sialic Acid Alpha 2,6 Sialidase Sialic acidHyperfucosylemia GalNAc Hexosaminidase GalNAc Hyperfucosylemia GalNAcSialidase Hexosaminidase GalNAc Hyperfucosylemia Sialic acidHexosaminidase Sialidase Sialic acid Hyperfucosylemia Galactosegalactosidase Galactose Hyperfucosylemia Galactose sialidasegalactosidase Galactose Hyperfucosylemia Fucose fucosidase FucoseHyperfucosylemia Galactose Galactosidase Galactose HyperfucosylemiaGlcNAc hexosaminidase GlcNAc Hyperfucosylemia Sulfate Sulfatase SulfateHyperfucosylemia Sulfated Sulfatase hexosaminidase GlcNAc or hexoseGalNAc Hyperfucosylemia Sulfated Sulfatase Iduronidase or IdoA or GlcAuronic acid glucouronidase Hypersulfogycanemia Sialic Acid SialidaseSialic acid Hypersulfogycanemia Sialic Acid Alpha 2,8 Sialidase Sialicacid Hypersulfogycanemia Sialic Acid Alpha 2,3 Sialidase Sialic acidHypersulfogycanemia Sialic Acid Alpha 2,6 Sialidase Sialic acidHypersulfogycanemia GalNAc Hexosaminidase GalNAc HypersulfogycanemiaGalNAc Sialidase Hexosaminidase GalNAc Hypersulfogycanemia Sialic acidHexosaminidase Sialidase Sialic acid Hypersulfogycanemia Galactosegalactosidase Galactose Hypersulfogycanemia Galactose sialidasegalactosidase Galactose Hypersulfogycanemia Fucose fucosidase FucoseHypersulfogycanemia Galactose Galactosidase GalactoseHypersulfogycanemia GlcNAc hexosaminidase GlcNAc HypersulfogycanemiaSulfate Sulfatase Sulfate Hypersulfogycanemia Sulfated Sulfatasehexosaminidase GlcNAc or hexose GalNAc Hypersulfogycanemia SulfatedSulfatase Iduronidase or IdoA or GlcA uronic acid glucouronidase

Provided herein are methods of diagnosing individuals (including, e.g.,a disease state or the severity of a disease states) with an infectiousdisease state associated with abnormal glycan accumulation. Provided inTable 15 are specific embodiments of disease that are optionallydiagnosed and/or monitored according to various embodiments describedherein. Table 15 also illustrates various non-limiting embodiments ofspecific enzyme(s) that are optionally utilized to treat a biologicalsample from an individual suffering from or suspected of (e.g., througha pre- or preliminary screening process) suffering from variousinfectious disease states associated with abnormal glycan accumulation.Moreover, Table 15 further illustrates various glycan residual compoundsthat are liberated in various embodiments described herein, suchliberated glycan residual compounds optionally being detected and/ormeasured in order to diagnose and/or monitor various infectious diseasestates.

TABLE 15 Infectious Diseases Primary Secondary Glycan Non-ReducingLiberating Liberating Residual Disease end structure Enzyme EnzymeCompound Bacterial Infections Mannose Mannosidase Mannose BacterialInfections Fucose Fucosidase Fucose Bacterial Infections GlucoseGlucosidase Glucose Bacterial Infections Galactose GalactosidaseGalactose Bacterial Infections GlcNAc hexosaminidase GlcNAc BacterialInfections GalNAc hexosaminidase GalNAc Bacterial Infections ArabinoseArabinosidase Arabinose Bacterial Infections Xylose Xylosidase XyloseBacterial Infections Ribose Ribosidase Ribose Bacterial InfectionsLyxose Lyxosidase Lyxose Bacterial Infections Talose Talosidase TaloseBacterial Infections Idose Idosidase Idose Bacterial Infections GuloseGulosidase Gulose Bacterial Infections Altrose Altrosidase AltroseBacterial Infections Allose Allosidase Allose Fungal Infections MannoseMannosidase Mannose Fungal Infections Fucose Fucosidase Fucose FungalInfections Glucose Glucosidase Glucose Fungal Infections GalactoseGalactosidase Galactose Fungal Infections GlcNAc hexosaminidase GlcNAcFungal Infections GalNAc hexosaminidase GalNAc Fungal InfectionsArabinose Arabinosidase Arabinose Fungal Infections Xylose XylosidaseXylose Fungal Infections Ribose Ribosidase Ribose Fungal InfectionsLyxose Lyxosidase Lyxose Fungal Infections Talose Talosidase TaloseFungal Infections Idose Idosidase Idose Fungal Infections GuloseGulosidase Gulose Fungal Infections Altrose Altrosidase Altrose FungalInfections Allose Allosidase Allose Viral Infections Sialic AcidSialidase Sialic acid Viral Infections Sialic Acid Alpha 2,8 Sialic acidSialidase Viral Infections Sialic Acid Alpha 2,3 Sialic acid SialidaseViral Infections Sialic Acid Alpha 2,6 Sialic acid Sialidase ViralInfections GalNAc Hexosaminidase GalNAc Viral Infections GalNAcSialidase Hexosaminidase GalNAc Viral Infections Sialic acidHexosaminidase Sialidase Sialic acid Viral Infections Galactosegalactosidase Galactose Viral Infections Galactose sialidasegalactosidase Galactose Viral Infections Fucose fucosidase Fucose ViralInfections Galactose Galactosidase Galactose Viral Infections GlcNAchexosaminidase GlcNAc Viral Infections Sulfate Sulfatase Sulfate ViralInfections Sulfated hexose Sulfatase hexosaminidase GlcNAc or GalNAcViral Infections Sulfated uronic Sulfatase Iduronidase or IdoA or GlcAacid glucouronidase

FIG. 1 illustrates compounds present in a normal biological sample notsubject to an enzymatic glycan residual liberation process describedherein. FIG. 2 illustrates compounds present in a normal biologicalsubject to an enzymatic glycan residual liberation process describedherein. FIG. 3 illustrates compounds present in a biological sample ofan individual suffering from a disorder associated with abnormal glycanaccumulation not subject to an enzymatic glycan residual liberationprocess described herein. FIG. 4 illustrates compounds present in abiological sample of an individual suffering from a disorder associatedwith abnormal glycan accumulation subject to an enzymatic glycanresidual liberation process described herein.

Detecting and Measuring:

Glycan residual compounds (including, e.g., oligosaccharides,monosaccharides, sulfate, phosphate, sialic acid, acetate, or the like)described herein are detected and/or measured in processes describedherein in any suitable manner. In some embodiments, glycan residualcompounds are detected and/or measured in unmodified form. In otherembodiments, glycan residual compounds are tagged with a detectablelabel prior and the labeled glycan residual compound is detected.

In some embodiments, non-labeled compounds are optionally detectedand/or measured in any suitable manner, e.g., by pH, by quantitativenuclear magnetic resonance (NMR), or the like.

In various embodiments, a method described herein comprises determiningwhether the amount of liberated glycan residue is abnormal and such adetermination comprises labeling the glycan residue with a detectablelabel and measuring the amount of labeled glycan residue with ananalytical instrument. In specific embodiments, the detectable label isa mass label, a radioisotope label, a fluorescent label, a chromophorelabel, or affinity label. In some embodiments, the amount of liberatedglycan is measured using UV-Vis spectroscopy, IR spectroscopy, massspectrometry, or a combination thereof.

In the various embodiments of any process or method described herein,any suitable detectable label is optionally utilized. In someembodiments, detectable labels useful in the processes or methodsdescribed herein include, by way of non-limiting example, mass labels,antibodies, affinity labels, radioisotope labels, chromophores,fluorescent labels, or the like.

Fluorescent labels suitable for use in various embodiments hereininclude, by way of non-limiting example, 2-aminopyridine (2-AP),2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminoacridone(AMAC), p-aminobenzoic acid ethyl ester (ABEE), p-aminobenzonitrile(ABN), 2-amino-6-cyanoethylpyridine (ACP), 7-amino-4-methylcoumarine(AMC), 8-aminonaphthalene-1,3,6-trisulfate (ANTS),7-aminonaphthalene-1,3-disulfide (ANDS), and8-aminopyrene-1,3,6-trisulfate (APTS), or the like. The fluorescentlabels can be attached by reductive amination with the fluorescent labeland sodium cyanoborohydride or the like.

Mass labels suitable for use in various embodiments herein include, byway of non-limiting example, D-2-anthranilic acid, D-2-aminopyridine,D-methyl iodide, ¹³C methyl iodide, deuterated-pyridyl-amine, D-biotinor the like. The mass labels can be attached by permethylation orreductive amination by any method that is known to those of skill in theart.

Affinity labels suitable for use in various embodiments herein include,by way of non-limiting example, biotin and derivatives.

Radioisotope labels suitable for use in various embodiments hereininclude, by way of non-limiting example, sodium borotritide (NaB³H₄),³H, ¹⁴C, ³²P, ³⁵S, or the like.

Chromophores suitable for use in various embodiments herein include, byway of non-limiting example, 4-amino-1,1′-azobenzene,4′-N,N-dimethylamino-4-aminoazobenzene, aminoazobenzene,diaminoazobenzene, Direct Red 16, CI Acid Red 57, CI Acid Blue 45, CIAcid Blue 22, CL Mordant Brown 13, CI Direct Orange 75, or the like. Thechromophores may be labeled by any method that is known to those ofskill in the art, such as reductive amination with the chromophore andsodium cyanoborohydride.

In some embodiments, the detectable label is an antibody. In specificembodiments, the antibody is attached to a detectable compound, such asmass labels, radioisotope labels, chromophores, fluorescent labels, orthe like. In some embodiments, antibodies are themselves detected and/orare detectable in various manners, e.g., as a chromophore, afluorophore, or the like; or with a probe (e.g., using dot blottechniques, immune-detection techniques, or the like).

In certain embodiments, detectable labels are detected and/or quantifiedaccording to any process described herein using any technique,particularly any technique suitable for the detectable label utilized.In some embodiments, suitable detection techniques include, by way ofnon-limiting example, one or more of a mass spectrometer, a nuclearmagnetic resonance spectrometer, a UV-Vis spectrometer, an IRspectrometer, a fluorimeter, a phosphorimeter, a radiation spectrometer(e.g., a scintillation counter), a thin layer chromatographic technique,or the like. In certain embodiments, in any process described herein,glycan residual compounds are optionally directly detected using asuitable technique, such as quantitative nuclear magnetic resonance.Quantitative nuclear magnetic resonance is also optionally utilized toquantify and/or detect the presence of a detectable label. In certainembodiments, one or more glycan residual compounds are optionallydetected using a suitable liquid chromatography mass spectrometer(LC-MS).

In some embodiments, glycan residual compounds are tagged with anantibody or probe, and are quantified using any suitable method (e.g.,dot blot techniques, immune detection techniques (e.g., ELISA), or thelike).

Various analytical methods useful for the processes described hereininclude, by way of non-limiting example, mass spectrometry,chromatography, HPLC, UPLC, TLC, GC, HPAEC-PAD,electrophoresis—capillary or gel, or the like. In certain embodiments,wherein a chromatographic technique is utilized, any suitable solventsystem is optionally employed. In certain embodiments, a column (e.g.,Cosmogel DEAE, Tsk Gel DEAE, Cosmogel QA, Cosmogel CM, Cosmogel SP, orthe like) is optionally loaded with an equilibrating solvent (e.g., abuffer or salt solution, such as a potassium acetate solution, sodiumchloride solution, sodium acetate solution, ammonium acetate solution,or the like), e.g., with a pH of about 6, 7, or 8. In some embodiments,the buffer or salt solution has a concentration of about 10 mM, 20 mM,30 mM, 50 mM, 100 mM, 500 mM, 1 M, 2 M, or the like. Any suitable flowrate is used, e.g., 0.5 mL/min, 1 mL, min, 1.5 mL/min, 2 mL/min, or thelike. Following equilibration, a linear gradient is optionally utilized.In some embodiments, the linear gradient is run over 1-20 min, 1-10 min,10-20 min, 1-5 min, 5-10 min, or the like. In certain embodiments, thegradient is a buffer or salt solution, e.g., as described above (e.g.,from 0 M to 0.5 M, from 0 M to 3 M, from 0.5 M to 2 M, from 0 M to 2 M,from 1M to 2M, from 0 M to 3 M, from 2 M to 0 M, from 3 M to 0 M, or thelike). Once the gradient has reached a final concentration, the eluentis optionally held at the final concentration for a suitable period oftime (e.g., 1-20 min, 5-10 min, 10-15 min, 1-5 min, 1-10 min, 15-20 min,or the like). After the optional holding of the final concentration, theeluent may be switched to a second solvent or solvent system (e.g., analcohol, such as methanol, ethanol, or isopropanol, acetonitrile, water,or the like). The switch to the second solvent system may be over aperiod of time, e.g., 15 seconds, 30 seconds, 45 seconds, 60 seconds, 2min, 3 min, or the like. The second solvent system is optionally heldfor a period of time, such as 1 min, 2 min, 3 min, 4 min, 5 min, 6 min,or the like. Following the second solvent system cycle, the column isoptionally restored to initial solvent conditions.

Purification:

In certain embodiments, methods described herein comprise purifying abiological sample, e.g., to remove non-glycan compounds from thebiological sample. In some embodiments, a biological sample is purifiedprior to transforming a glycan thereof.

In certain embodiments, a biological sample containing glycans (purifiedor not) can also be prepared so that all free glycan residual compounds(e.g., monosaccharides) that are naturally present in the biologicalsample (i.e., as taken from an individual and without being treated) areeliminated from the sample to reduce background signal (for exampleusing dialysis, spin column, gel filtration, etc).

In some embodiments, any process described herein includes a step ofpurifying a biological sample comprising removing monosaccharidestherefrom, removing sulfates therefrom, removing phosphates therefrom,removing acetate therefrom, removing sialic acid therefrom, or acombination thereof. For example, in some embodiments, a biologicalsample is optionally placed in to a defined MW cut off spin column(retains large molecules when spun), optionally washed (e.g., with 1 ormore volumes of water or buffer), and/or the like.

In certain embodiments, purification of biological samples may furtheror alternatively comprise, e.g., fractionation, purification,enrichment, or the like of glycans contained therein. In some instances,such purification techniques are suitable to isolate and/or separatedifferent glycan classes within the biological sample prior totransformation of one or more of such glycans. In more specificinstances, such purification techniques are used to isolate and/orseparate different subsets of a single glycan class (such as isolatingcomplex N-linked glycans from hybrid N-linked structures) prior totransformation of one or more of such glycans. In certain embodiments, abiological sample is optionally prepared in such a way to enrich forspecific glycan classes. For example, a PHA affinity column isoptionally used to isolate a sub-fraction of complex N-linked glycanswhile a Con A column could be used to enrich in a different subset ofN-linked glycans.

In some embodiments, any process described herein comprises purificationof a glycan residual compound resulting from a process described herein(e.g., purification of the glycan residual compound prior to analysisthereof). For example, in some embodiments, the glycan residual compoundis optionally isolated by any suitable process, such as by washing thefree glycan residual compound (e.g., through a defined MW cut offmembrane or by any other suitable method). Moreover, in certainembodiments, the resulting isolated glycan residual compound containingcomposition is optionally dried or otherwise treated to concentrate thesample and subsequently analyzed for glycan residual compound content byany suitable analytical technique.

In some embodiments, the processes described herein comprises furthertreatment steps of the test and/or control samples. For example, in someembodiments, the samples are homogenized and/or purified. In specificembodiments homogenization is achieved in any suitable manner including,by way of non-limiting example, with a basic solution, sonication,tissue grinding, or other chemical agents. In some embodiments, severityof a disorder is determined if a certain threshold amount is measured(e.g., as compared to a control or controls) or a threshold signal(e.g., on a fluorimeter or other analytical device utilized to detectand/or measure the generated biomarker). Similarly, a carrier of adisorder described herein is, in certain embodiments, determined if acertain threshold amount is measured (e.g., as compared to a control orcontrols) or a threshold signal (e.g., on a fluorimeter or otheranalytical device utilized to detect and/or measure the generatedbiomarker).

In certain embodiments, samples, including test samples and/or controlsamples, described herein are optionally purified prior to glycanprocessing (e.g., lyase treatment) and/or characterization. Test samplesand/or control samples (i.e., one or more or all of the glycans foundtherein) are optionally purified using any suitable purificationtechnique. Test samples and/or control samples are optionally purifiedat any suitable point in a process described herein, including before orafter tagging of the glycans founds within the sample. In certainembodiments, purification techniques include centrifugation,electrophoresis, chromatography (e.g., silica gel or alumina columnchromatography), gas chromatography, high performance liquidchromatography (HPLC) (e.g., reverse phase HPLC on chiral or achiralcolumns), thin layer chromatography, ion exchange chromatography, gelchromatography (e.g., gel filtration or permeation or size exclusionchromatography, gel electrophoresis), molecular sieve chromatography,affinity chromatography, size exclusion, filtration (e.g. through aflorisil or activated charcoal plug), precipitation, osmosis,recrystallization, fluorous phase purification, distillation,extraction, chromatofocusing, supercritical fluid extraction,preparative flash chromatography (e.g., flash chromatography using aUV-Vis detector and/or a mass spectrometer (e.g., using the Biotage®suite of products) or the like.

In some embodiments, glycans, such as heparan sulfate, are naturallyfound attached to a core protein (together forming a proteoglycan) or alipid. In some embodiments, provided herein are purification processesof separating glycan fragments (e.g., heparan sulfate fragments) fromproteoglycans or glycolipids prior to processing the glycan forprocessing and analysis.

Monitoring Therapy

Provided in certain embodiments are methods of treating disordersassociated with the abnormal degradation, biosynthesis and/oraccumulation of glycans, the methods comprising:

-   -   a. administering an agent for treating disorders associated with        the abnormal degradation, biosynthesis and/or accumulation of        glycans (e.g., an anti-LSD agent, an anti-cancer agent, or the        like) to an individual in need thereof;    -   b. monitoring the accumulation of glycans in the individual        using any process described herein for detecting or quantifying        the amount of glycan residual compounds (e.g., mono-saccharides,        sulfate, or the like) present in a lyase digested biological        sample (e.g., urine, serum, plasma, or CSF sample) according to        any process described herein.

Provided in further or alternative embodiments are methods of monitoringthe treatment of disorders associated with the abnormal degradation,biosynthesis and/or accumulation of glycans, the methods comprising thefollowing steps:

-   -   a. following administration of an agent for treating a disorder        associated with the abnormal degradation, biosynthesis and/or        accumulation of glycans (e.g., an anti-LSD agent, an anti-cancer        agent, or the like) to an individual in need thereof, generating        a biomarker comprising of one or more non-reducing end glycan        residual compound (e.g., monosaccharide).

In some embodiments, the biomarker is a saturated monosaccharide and isgenerated by treating a population of glycans, in or isolated from abiological sample from the individual, with at least one digestingglycan enzymes, wherein prior to enzyme treatment, the biomarker is notpresent in abundance in samples from individuals with the disease orcondition relative to individuals without the disease or condition. Incertain embodiments, monitoring of the accumulation of glycans comprisesusing an analytical instrument to detect the presence of and/or measurethe amount of the biomarker produced and displaying or recording thepresence of or a measure of a population of the biomarker; wherein thepresence of and/or measure the amount of the biomarker is utilized tomonitor the treatment.

In some embodiments, the agent is administered one or more times. Incertain embodiments, the agent is administered multiple times. In someembodiments, the agent is administered in a loading dose one or moretimes (e.g., in a loading dosing schedule) and subsequently administeredin a maintenance dose (e.g., in a maintenance dosing schedule, such asthree times a day, twice a day, once a day, once every two days, onceevery three days, once every four days, once a week, or the like). Insome embodiments, when glycan (as measure by one or more glycan residualcompound(s)) accumulation begins to increase or accelerate, the dose isoptionally adjusted (e.g., the maintenance dose is increased, or anadditional loading dose or dosing schedule is utilized).

In some embodiments, monitoring the accumulation of glycans comprisesrepeating the step of: using an analytical instrument to detect thepresence of and/or measure the amount of a population of one or moreglycan residual compounds present in a transformed biological samplethat has been prepared by treating a population of glycans, in orisolated from a biological sample from the individual, with at least onedigesting glycan lyase to transform the glycan into the population ofthe one or more glycan residual compounds. In specific embodiments, thestep is repeated at periodic intervals (e.g., every day, every otherday, every 2 days, every 3 days, every 4 days, every week, every month,every 3 months, quarterly, every 6 months, yearly, or the like), atregular times following a dose (e.g., 4 hours after a administration ofthe agent, 6 hours after administration of the agent, 8 hours afteradministration of the agent, 12 hours after administration of the agent,or the like), prior to administration of the dose (e.g., immediatelyprior to administration of the agent, 2 hours prior to administration ofthe agent, or the like), or any other monitoring schedule.

In some embodiments, the monitoring of the accumulation of glycan isconducted over a period of time, e.g., over a week, two weeks, a month,two months, three months, six months, a year, or the like. In someembodiments, the method for quantifying the amount of one or more glycanresidual compounds in a lyase digested biological sample (e.g., urine,serum, plasma, or CSF) comprises detecting and/or measuring (e.g., withan analytical device), one or more glycan residual compounds within thelyase digested biological sample from the individual after thebiological sample obtained from the individual has been treated with oneor more glycan lyases. In certain embodiments, such glycan lyases aresuitable for preparing glycan residual compounds from the glycan presentin the biological sample obtained from the individual. In certaininstances a representative portion of the one or more glycan residualcompounds in the transformed biological sample is tagged with anysuitable detectable label (e.g., a mass label, a radioisotope label, afluorescent label, a chromophore label, affinity label, an antibody). Insome embodiments, the process comprises displaying or recording such acharacterization of the population of glycan residual compounds and/ortagged glycan residual compounds.

In some embodiments, the agent described in a therapy herein includesglycan accumulation inhibitors, agents that promote glycan degradation,agents that activate enzymes that degrade glycans, agents that inhibitbiosynthesis of glycans, or the like. In some embodiments, the agentthat modulates glycan biosynthesis is an agent that selectivelymodulates heparan sulfate biosynthesis, an agent that selectivelymodulates chondroitin sulfate biosynthesis, an agent that selectivelymodulates dermatan sulfate biosynthesis, an agent that selectivelymodulates keratan sulfate biosynthesis, an agent that selectivelymodulates hyaluronan biosynthesis, or a combination thereof. Anti-LSDdrugs include, by way of non-limiting example, Imiglucerase (Cerazyme),laronidase (Aldurazyme), idursulfase (Elaprase), galsulfase (Naglazyme),agalsidase beta (Fabrazyme), alglucosidase alfa (Myozyme), agalsidasealfa (Replagal), miglustat (Zavesca).

In some embodiments, one or more of the anti-cancer agents areproapoptotic agents. Examples of anti-cancer agents include, by way ofnon-limiting example: gossyphol, genasense, polyphenol E, Chlorofusin,all trans-retinoic acid (ATRA), bryostatin, tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL),5-aza-2′-deoxycytidine, all trans retinoic acid, doxorubicin,vincristine, etoposide, gemcitabine, imatinib (Glcevec®), geldanamycin,17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol,LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, or PD184352,Taxol™, also referred to as “paclitaxel”, which is a well-knownanti-cancer drug which acts by enhancing and stabilizing microtubuleformation, and analogs of Taxol™, such as Taxotere™. Compounds that havethe basic taxane skeleton as a common structure feature, have also beenshown to have the ability to arrest cells in the G2-M phases due tostabilized microtubules and may be useful for treating cancer incombination with the compounds described herein.

Further examples of anti-cancer agents include inhibitors ofmitogen-activated protein kinase signaling, e.g., U0126, PD98059,PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006,wortmannin, or LY294002; Syk inhibitors; mTOR inhibitors; and antibodies(e.g., rituxan).

Other anti-cancer agents include Adriamycin, Dactinomycin, Bleomycin,Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride;acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantroneacetate; aminoglutethimide; amsacrine; anastrozole; anthramycin;asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine;gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride;ifosfamide; iimofosine; interleukin Il (including recombinantinterleukin II, or rlL2), interferon alfa-2a; interferon alfa-2b;interferon alfa-n1; interferon alfa-n3; interferon beta-1 a; interferongamma-1 b; iproplatin; irinotecan hydrochloride; lanreotide acetate;letrozole; leuprolide acetate; liarozole hydrochloride; lometrexolsodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride.

Other anti-cancer agents include: 20-epi-1, 25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron;doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen;ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur;epirubicin; epristeride; estramustine analogue; estrogen agonists;estrogen antagonists; etanidazole; etoposide phosphate; exemestane;fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

Yet other anticancer agents that include alkylating agents,antimetabolites, natural products, or hormones, e.g., nitrogen mustards(e.g., mechloroethamine, cyclophosphamide, chlorambucil, etc.), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne,ete.), or triazenes (decarbazine, etc.). Examples of antimetabolitesinclude but are not limited to folic acid analog (e.g., methotrexate),or pyrimidine analogs (e.g., Cytarabine), purine analogs (e.g.,mercaptopurine, thioguanine, pentostatin).

Examples of natural products include but are not limited to vincaalkaloids (e.g., vinblastin, vincristine), epipodophyllotoxins (e.g.,etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin),enzymes (e.g., L-asparaginase), or biological response modifiers (e.g.,interferon alpha).

Examples of alkylating agents include, but are not limited to, nitrogenmustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil,meiphalan, etc.), ethylenimine and methylmelamines (e.g.,hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan),nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin,etc.), or triazenes (decarbazine, ete.). Examples of antimetabolitesinclude, but are not limited to folic acid analog (e.g., methotrexate),or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine),purine analogs (e.g., mercaptopurine, thioguanine, pentostatin.

Examples of hormones and antagonists include, but are not limited to,adrenocorticosteroids (e.g., prednisone), progestins (e.g.,hydroxyprogesterone caproate, megestrol acetate, medroxyprogesteroneacetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol),antiestrogen (e.g., tamoxifen), androgens (e.g., testosteronepropionate, fluoxymesterone), antiandrogen (e.g., flutamide),gonadotropin releasing hormone analog (e.g., leuprolide). Other agentsthat can be used in the methods and compositions described herein forthe treatment or prevention of cancer include platinum coordinationcomplexes (e.g., cisplatin, carboblatin), anthracenedione (e.g.,mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazinederivative (e.g., procarbazine), adrenocortical suppressant (e.g.,mitotane, aminoglutethimide).

In some instances, the detection and/or the quantification of theidentity and/or amount of glycan residual compounds present in abiological sample is used to identify and/or diagnose a disorderassociated with abnormal degradation, biosynthesis and/or accumulationof glycan in an individual suspected of having such a disorder.

In some instances, the detection and/or the quantification of theidentity and/or amount of glycan residual compounds present in thebiological sample is used to monitor severity and course of the diseasein an individual diagnosed with or suspected of having a disorderassociated with the abnormal degradation, biosynthesis and/oraccumulation of glycans. In some instances, the detection and/or thequantification of the identity and/or amount of glycan residualcompounds present in the biological sample is used to calculate theadministered dose of an agent that modulates (e.g., promotes and/orinhibits) glycan biosynthesis and/or degradation.

In certain instances, wherein following administration of a selecteddose of a therapeutic agent utilized in a therapeutic method describedherein, an individual's condition does not improve, the detection and/orthe quantification of the identity and/or amount of glycan residualcompounds present in a biological sample provides for a treatmentregimen to be modified depending on the severity and course of thedisease, disorder or condition, previous therapy, the individual'shealth status and response to the drugs, and the judgment of thetreating physician.

In certain embodiments, monitoring the accumulation of glycans in theindividual comprises detecting or quantifying the amount of an glycanresidual compounds (or one or more glycan residual compounds) in asample obtained from the individual (e.g., according to any methoddescribed herein) to obtain a first accumulation result (e.g., aninitial reading before treatment has begun, or at any other time) and asecond accumulation result that is subsequent to obtaining the firstresult. In some embodiments, the second result is compared to the firstresult to determine if the treatment is effectively reducing,maintaining, or reducing the rate of increasing the glycan residualcompounds levels in a substantially identically obtained sample from theindividual being treated. In certain embodiments, depending on thedifference between the first and second results, the treatment can bealtered, e.g., to increase or decrease the amount of agent administered;to substitute the therapeutic agent with an alternative therapeuticagent; or the like. In certain embodiments, the dose of the therapeuticagent is decreased to a maintenance level (e.g., if the glycan residualcompound level has been reduced sufficiently); further monitoring ofglycan residual compound levels is optional in such situation, e.g., toensure that reduced or maintained levels of glycan residual compounds(e.g., monosaccharide(s)) are achieved.

Alternatively, provided herein is a method of detecting response totherapy in an individual or a method of predicting response to therapyin an individual comprising:

-   -   a. administering an agent for treating a disorder associated        with the abnormal degradation, biosynthesis and/or accumulation        of glycans to a plurality of cells from an individual in need        thereof (e.g., a plurality of fibroblasts, serum, plasma, or CSF        cells from a human suffering from a disorder associated with the        abnormal degradation, biosynthesis and/or accumulation of        glycans, such as an LSD or cancer);    -   b. monitoring the accumulation of glycans in the plurality of        cells using any process described herein for detecting or        quantifying the amount of glycan residual compounds (e.g.,        monosaccharides, sulfate, sialic acid, phosphate, acetate, or        the like) present in a lyase digested biological sample from the        plurality of cells according to any process described herein.

In specific embodiments, the glycan residual compound(s) detected ormeasured is one or more monosaccharide. It is to be understood that aplurality of cells from an individual includes cells that are directlytaken from the individual, and/or cells that are taken from anindividual followed by culturing to expand the population thereof.

EXAMPLES Example 1

To illustrate the methods described herein, we have used human urinesample from normal patients and patients diagnosed with MPS IIIA. MPSIIIA patients have reduced function of the lysosomal enzyme thatde-N-sulfates the nonreducing end glucosamine residues present inheparan sulfate. This unique nonreducing end glycan residual (N-sulfatedGlcN) can be liberated by treating the glycans with heparin lyases andquantified by fluorescent detection on HPLC. As shown below, glycansprepared in this manner from normal individuals lack N-sulfate GlcNwhile MPS IIIA patients have a very high level.

Purification: The biological sample (cells, tissue, blood, serum, or thelike) is homogenized and solublized in 0.1-1.0 N NaOH (e.g., 0.1 N, 0.2N, 0.3 N, 0.4 N, 0.5 N, 0.6 N, 0.7 N, 0.8 N, 0.9 N, or 1.0 N) or aceticacid and then neutralized with acetic acid or NaOH. Next a small sampleis taken to measure protein content of the sample using standardmethods. 0.01-0.5 mg/mL (0.01 mg/mL, 0.07 mg/mL, 0.12 mg/mL, 0.17 mg/mL,0.22 mg/mL, 0.27 mg/mL, 0.32 mg/mL, 0.37 mg/mL, 0.42 mg/mL, or 0.5mg/mL) protease (trypsin, chymotrypsin, pepsin, pronase, papain, orelastase) is treated in 0.1-0.5 M (e.g., 0.1 M, 0.16 M, 0.23 M, 0.32 M,0.39 M, 0.44 M, or 0.5 M) NaCl, 0.01-0.1 M (e.g., 0.01 M, 0.02 M, 0.04M, 0.06 M, 0.08 M, 0.1 M) NaOAc, at pH 5.5-7.5 (e.g., 5.5, 6.0, 6.5,7.0, or 7.5) and 25-40 C (e.g., 25 C, 30 C, 35 C, or 40 C) for 1-24hours (e.g., 1 h, 2 h, 4 h, 6 h, 8h, 12 h, 18 h, 24 h). The sample isdiluted to reduce the ionic strength and loaded onto an ion exchangecolumn in 5-100 mM (e.g., 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60mM, 70 mM, 75 mM, 80 mM, 90 mM, 95 mM, 100 mM) NaOAc pH 5-7 with 0-300mM NaCl. After washing, the bound glycosaminoglycans are eluted with5-100 mM NaOAc pH 5-7 (e.g., 5, 5.5, 6, 6.5, 7) with 0.8-3 M (e.g., 0.8M, 1 M, 1.2 M, 1.4 M, 1.6 M, 1.8 M, 2 M, 2.5 M, or 3 M) NaCl. The elutedglycans are then concentrated and desalted by ethanol precipitation,size exclusion, or other methods. The purified glycans are dried forfurther analysis.

Liberation of non-reducing end residual: The purified glycans areresuspended in 10-300 mM sodium acetate, tris, phosphate, or othersuitable buffer, 0.02-1 mM (e.g., 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1) calcium acetate, pH 5-8 (e.g., 5,5.5, 6, 6.5, 7, 7.5, or 8), were digested with heparin lyases I, II,III, I and II, I and III, II and III, or I, II, and III (0.0.15-1.5milliunits of each in 100-ul reactions, IBEX, Montreal, Canada) at 25 to37° C. for 1 to 24 hours.

Fluorescent tagging of glycan residual: Dried glycan sample isre-suspended in 2-100 0.003-0.1 M (e.g., 0.003 M, 0.003 M, 0.03 M, 0.06M, 0.1 M) AB, AA, AMAC, or Bodipy dye and incubated at room temperaturefor 1-120 minutes (e.g., 1-10 min, 10-15 min, 15-20 min, 20-25 min,25-30 min, 30-40 min, 40-50 min, 50-60 min, 60-90 min, 90-120 min).Next, the reaction is initiated with 2-100 μL (2 μL, 5 μL, 10 μL, 15 μL,20 μL, 25 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, or 100μL) 1 M NaCNBH₄ and the reaction is allowed to proceed at 25-100 C.(e.g., 25 C, 30 C, 35 C, 40 C, 50 C, 60 C, 70 C, 80 C, 90 C, 100 C).

Detection of glycan residual: HPLC separation of tagged saccharides wasperformed utilizing the following conditions: Column types: 130A BEHparticle Phenyl (1.7, 2.5, 3.5, 5, or 10 uM particle size), 130A BEHparticle C18 (1.7, 2.5, 3.5, 5, or 10 uM particle size), HSS particleC18 (1.8, 3.5, or 5 uM particle size), or 300A BEH particle C18 (1.7,3.5, 5, 10 uM particle size) with suitable length and internal diameter.

Buffer Conditions:

A=Ammonium Acetate, Sodium Acetate, or Sodium Chloride (e.g., 0 M, 10mM, 20 mM, 30 mM, 40 mM, 100 mM, 500 mM, 1 M, 2 M) with 0-20% methanolB=100% Alcohol, such as methanol, ethanol, or isopropanol

Initial Conditions: 70-95% A, 0-30% B

Flow Rate is constant at 0.05-1 ml/minRuns a gradient down to 70-90% A, 10-30% B over 5-65 min.At 8.1 min runs a gradient to 0-20% A, 80-100% B over 5-20 min.5-65 min returns to initial conditions

FIG. 1 illustrates an HPLC trace of eluted compounds detected in normalpatient urine not subject to enzymatic glycan residual liberation (i.e.,providing background signals). FIG. 2 illustrates an HPLC trace ofeluted compounds detected in normal patient urine subject to enzymaticglycan residual liberation as set forth in Example 1. FIG. 3 illustratesan HPLC trace of eluted compounds detected in MPS IIIA patient urine notsubject to enzymatic glycan residual liberation (i.e., providingbackground signals). FIG. 4 illustrates an HPLC trace of elutedcompounds detected in MPS IIIA patient urine subject to enzymatic glycanresidual liberation.

Example 2

The processes described in Example 1 are repeated and/or modified forthe diseases listed in Tables 1-4 utilizing the enzymes described therein and detecting the glycan residual compounds also described therein.

Example 3: NRE Analysis of MPS I, II, VI and VII Cells

To demonstrate the potential utility of this approach, dermalfibroblasts from human MPS I patients (α-iduronidase [IDUA] deficiency)and from normal human donors were grown. Cells were expanded and kept inculture up to 8 weeks to allow for significant lysosomal accumulation.GAGs remaining in the cell layer were extracted and subjected toenzymatic depolymerization followed by reductive amination with[¹²C₆]aniline. Samples were mixed with 10 pmoles of each [¹³C₆]anilineunsaturated disaccharide standard and [¹³C₆]aniline-tagged I0S0 that wassynthesized. All possible candidate structures were searched for (FIG.6) and the extracted ion chromatogram shown in FIG. 7a . Peaks 1-7comigrated with known unsaturated disaccharides and had the expected m/zvalues. The MPS I sample had an additional peak marked by an asteriskthat was not present in the normal fibroblast sample. This peak had thesame elution position as the aniline-tagged I0S0 standard (expandedinset in FIG. 7a ). The mass spectrum for this peak gave an m/z=511.1and isotopic cluster consistent with the proposed structure I0S0, and acorresponding m/z=517.1 and isotopic cluster expected for the[¹³C₆]aniline tagged standard (FIG. 7b ). Further verification wascarried out by collision-induced dissociation (CID), which demonstratedstructural identity with the I0S0 standard (FIG. 10). Digestion ofheparan sulfate from cells derived from MPS I patients also yielded adisaccharide of m/z=591.1 (FIG. 11a ), consistent with the structure10S6. This material comigrated with the internal disaccharide D2S0 andthus was contained within peak 6 in the chromatogram shown in FIG. 7a .However, it was easily discriminated from D2S0 by the mass detectorgiven the 18 amu difference. Pretreatment of an MPS I sample withα-L-iduronidase led to the loss of the native NRE structures confirmingtheir identity (FIG. 12). Fibroblasts from three different MPS Ipatients exhibited NRE species identified as I0S0 and 10S6. Theseentities were not observed in samples prepared from normal humanfibroblasts.

MPS II (idurono-2-sulfatase [IDS] deficiency) and MPS VII(β-D-glucuronidase [GLCA] deficiency) also affect heparan sulfatedegradation due to defects in processing the non-reducing terminaluronic acid (desulfation of iduronate-2-sulfate and removal ofglucuronic acid, respectively). Saturated NRE disaccharides weredetected after digestion of GAGs derived from fibroblasts of MPS II andMPS VII patients (FIG. 6 and FIGS. 11b and 11c ). The mass spectra forthe MPS II NREs and their elution positions were consistent with theexpected disaccharide biomarkers I2S0 and I2S6. Analysis of MPS VIIheparan sulfate was more complex, yielding four disaccharidestentatively identified as G0A0, G0A6, G0S0 and G0S6. Treatment of theMPS II samples with recombinant IDS converted the NRE to those found inMPS I (I0S0 and 10S6, respectively; FIG. 13).

MPS I, MPS II, and MPS VII also affect the degradation of chondroitinsulfate and dermatan sulfate. Analysis of these GAGs usingchondroitinase ABC yielded a set of NRE disaccharides diagnostic foreach disorder (FIG. 6). MPS I yielded the monosulfated NRE disaccharidesI0a4 and I0a6 in addition to the disulfated disaccharide I0a10 (FIG. 11d). MPS II yielded I2a4 and I2a6 (FIG. 11e ) and MPS VII yielded G0a0,G0a4, G0a6 and G0a10 (FIG. 11f ).

Analysis of chondroitin sulfate and dermatan sulfate from MPS VI(N-acetylgalactosamine 4-sulfatase [G4S] deficiency) demonstratedaccumulation of N-acetylgalactosamine-4-sulfate (a4, FIG. 6), whichco-migrated with an aniline tagged a4 standard (FIG. 14). Note that a4resolves partially by liquid chromatographically from6-sulfo-N-acetylgalactosamine (a6) (lower panel). However, thebiological sample yielded predominantly a4, consistent with thedeficiency in the N-acetylgalactosamine 4-sulfatase in these cells. Notrisaccharides species were detected.

Example 4: NRE Analysis of MPS III Cells

The Sanfilippo family of MPS disorders was analyzed using the sameapproach as above: MPS IIIA (sulfamidase [SGSH] deficiency), MPS IIIB(α-N-acetylglucosaminidase [NAGLU] deficiency), MPS IIIC(N-acetyltransferase [HGSNAT] deficiency) and MPS IIID(glucosamine-6-sulfatase [GNS] deficiency). These disorders only affectlysosomal degradation of heparan sulfate and have in common deficienciesin the enzymes that process the NRE glucosamine residue. Because heparinlyases cleave linkages between a glucosamine unit and a uronic acid, itwas expected that analysis of Sanfilippo heparan sulfate should yielddiagnostic monosaccharides (glucosamine derivatives) or trisaccharides(glucosamine-uronic acid-glucosamine derivatives) from the NRE asopposed to the disaccharides observed in MPS I, II and VII.

Analysis of MPS IIIA samples showed the typical unsaturateddisaccharides generated from internal segments of the chains and aunique peak at 38.5 minutes not present in control or other MPS samples.This material had the characteristic mass spectrum expected for[¹²C₆]aniline-tagged N-sulfoglucosamine (S0, m/z=335.1) and comigratedwith an authentic [¹³C₆]aniline-tagged standard (m/z=341.1; FIG. 7c ,inset). Treatment of two different MPS IIIA samples with sulfamidaseprior to heparinase depolymerization destroyed the S0 biomarker,consistent with its proposed identity (FIG. 15). Digestion of MPS IIIAheparan sulfate also yielded trisaccharides that varied in the number ofacetate and sulfate groups (FIG. 7c , dp3). The most prominent speciesdp3(0Ac,3S) was analyzed by CID and gave a fragmentation patternconsistent with S0U2S0 (FIG. 16a ). Although the uronic acid could beglucuronic acid, iduronic acid predominates in segments of the chaincontaining repeating N-sulfoglucosamine units.

Analysis of MPS TIM samples yielded three NRE trisaccharides, with m/zvalues consistent with the presence of 1-2 acetate groups and 1-3sulfates (FIG. 7d ). Since MPS TIM is characterized by the lack ofα-N-acetylglucosaminidase, the terminal sugar should beN-acetylglucosamine, which was confirmed by CID analysis of thepredominant trisaccharide (m/z=794) identified as A0U2S0 (FIG. 16b ).Similarly, the NREs from MPS IIIC were predicted to contain a freeunsubstituted amine due to the deficiency of glucosamineN-acetyltransferase. Four trisaccharides were detected, all lackingacetate groups (FIG. 7e ). CID analysis of dp3(0Ac,3S) andderivatization with propionyl anhydride suggested structures consistentwith H6U0S6 or H0U2S6 (FIG. 16c ).

MPS IIID cells lack the 6-sulfatase that can remove the 6-O-sulfategroup from terminal N-acetylglucosamine units. NRE analysis of MPS IIIDheparan sulfate detected a single monosaccharide species with m/z valueof 335 corresponding to N-unsubstituted GIcNH26S (H6) (FIG. 7f ). WhileH6 is isobaric with S0 found in MPS IIIA, its retention time wassignificantly less due to the presence of the unsubstituted amine andconsequently these markers were easily discriminated. Furthermore, H6 inMPS IIID co-eluted with [¹³C₆]aniline-labeled standard H6 verifying itsidentity (FIG. 7f , inset). No N-acetylglucosamine-6-sulfate wasdetected, nor were any trisaccharide NRE species bearing a non-reducingterminal 6-O-sulfated N-acetylglucosamine unit (FIG. 70.

Example 5: Use of NRE Biomarkers

Although most MPS GAG samples yielded multiple NRE carbohydrates (FIG.6), it was possible to select single unique NREs as biomarkers for eachMPS disorder and then combine them into a decision tree based on size ofNRE structures (mono-, di- and trisaccharides), degree of sulfation, andretention time during liquid chromatography (FIG. 8). The diagnosticdecision tree becomes even more robust by inclusion of othercarbohydrate biomarkers (FIG. 6), but the specific NREs indicated inFIG. 8 are sufficient to diagnose the eight MPS disorders. To determinethe potential utility of these markers for diagnosis, nine differenthuman urine samples from normal control subjects and patients sufferingfrom various Sanfilippo disorders were screened as well as two canineurine samples (one normal and one with MPS I) and liver, brain andkidney GAGs from MPS IIIB mice. Using the scheme outlined in FIG. 8 allsamples were correctly diagnosed (Table 16).

Table 16. Analysis of GAG samples purified from mouse tissues and humanand canine urine. GAG was extracted and analyzed for MPS diagnosticbiomarkers using the scheme shown in FIG. 8. Diagnostic markers: MPS I,I0S0; MPS II, I2S6; MPS IIIA, S0 and S0U250; MPS IIIB, A0U2S0; MPS IIICdp3(0Ac,3S0₄), a trisaccharide containing no acetate groups and threesulfate groups; MPS IIID, H6; MPS VI, a4; MPS VII, G0S0.

TABLE 16 NRE Biomarkers Sensi-Pro Sample Sample Identity DetectedAnalysis Liver, Mouse-1 Unaffected, MPS IIIB Trace Normal Carrier (Het)Liver, Mouse-2 MPS IIIB A0U2S0 MPSIIIB Brain, Mouse-1 Unaffected, MPSIIIB Trace Normal Carrier (Het) Brain, Mouse-2 MPS IIIB A0U2S0 MPSIIIBKidney, Mouse-1 MPS IIIB A0U2S0 MPSIIIB Urine, Human-1 Normal TraceNormal Urine, Human-2 Normal Trace Normal Urine, Human-3 Normal TraceNormal Urine, Human-4 Normal Trace Normal Urine, Human-5 MPS IIICdp3(0Ac,3S0₄) MPSIIIC Urine, Human-6 MPS IIIC dp3(0Ac,3S0₄) MPSIIICUrine, Human-7 MPS IIIC dp3(0Ac,3S0₄) MPSIIIC Urine, Human-8 MPS IIIAS0, S0U2S0 MPSIIIA Urine, Human-9 MPS IIIB A0U2S0 MPSIIIB Urine,Canine-1 Unaffected, MPS I Trace Normal Carrier (Het) Urine, Canine-2MPS I I0S0 MPS I

Analysis of multiple MPS IIIA cell lines showed striking accumulation ofthe S0 biomarker, which corresponded well with the level of heparansulfate storage (Table 17). Normal fibroblasts yielded minute amounts ofS0. In general, samples from normal cells, tissues and urine exhibitedless than 1% of the amount of NRE biomarkers observed in samples fromaffected patients or animals.

TABLE 17 Quantitation of markers in MPS IIIA cells and normalfibroblasts Enzyme activity Heparan Sulfate MPS IIIA marker: S0 Cellline (Units/mg) (nmol/mg cell protein) (pmol/mg cell protein) Normal   9± 0.7 0.59 ± 0.22 2 ± 2 GM00629 0.49 ± 0.12 33.6 670 GM00643 0.45 ± 0.0828.4 720 GM00879 0.62 ± 0.02 17.3 490 GM00934 0.46 ± 0.07 16.6 470GM06110 0.55 ± 0.36  6.8 220

Five normal fibroblasts were analyzed (CRL-1634 (human foreskinfibroblasts), GM00200 (clinically unaffected sibling of metachromaticleukodystrophy patient), GM05659, GM08398, and GM15871 (clinicallyunaffected sibling of an Ehlers-Danlos patient). The averagevalues±standard deviation are provided. The values for the various MPSIIIA lines represent duplicate analyses for enzyme activity and singledeterminations for heparan sulfate storage and the S0 biomarker.

The detection of lysosomal storage based on GAG content in the brain andurine has been challenging due to various methods used for detection andquantitation, in particular indirect techniques based on dye binding ordisplacement. Urine and brain samples from MPS IIIA (Sgsh^(−/−)) andwildtype mice and MPS IIIA human fibroblasts were analyzed using thescheme described in FIG. 8. Using this method, total heparan sulfate andthe biomarker S0 showed a 12-fold accumulation in MPS IIIA urine samplescompared to the wildtype (FIG. 9a ). The trisaccharide biomarker S0U2S0was readily detectable in the Sgsh^(−/−) urine, but virtuallyundetectable in wildtype urine. In brain samples the heparan sulfatelevel was elevated 12-fold, whereas the biomarker S0 increased 60-foldcompared to the wildtype (FIG. 9b ). The trisaccharide marker wasessentially present only in the Sgsh^(−/−) sample.

In order to test whether the NRE structures afford a more precise andsensitive assay to monitor therapeutic enzyme replacement, cultures ofMPS IIIA human fibroblasts were supplemented with recombinantsulfamidase for 48 hours prior to GAG extraction and analysis. Enzymereplacement led to a significant drop in heparan sulfate and both thebiomarkers, S0 and S0U2S0 (FIG. 9c ). Thus, the NRE biomarkers, inparticular the trisaccharides, are useful for monitoring the therapeuticefficacy of intervention strategies.

Example 6: Synthesis of I0S0

All moisture sensitive reactions were performed under an argonatmosphere by using vacuum dried glassware. All commercial materialswere used without purification, unless otherwise noted. CH₂Cl₂ wasfreshly distilled from calcium hydride under nitrogen prior to use.Toluene, DMF, diethylether, methanol and THF were purchased anhydrousand used without further purification. Molecular sieves (4 Å) were flameactivated in vacuo prior to use. All reactions were performed at roomtemperature unless specified otherwise. TLC analysis was conducted onSilica gel 60 F254 (EMD Chemicals Inc.) with detection by UV-absorption(254 nm) when applicable, and by spraying with 20% sulfuric acid inethanol followed by charring at ˜150° C. or by spraying with a solutionof (NH₄)₆M_(O7)O₂₄H₂O (25 g/L) in 10% sulfuric acid in ethanol followedby charring at ˜150° C. Column chromatography was performed on silicagel G60 (Silicycle, 60-200 μm, 60 Å) or on Bondapak C-18 (Waters). ¹Hand ¹³C NMR spectra were recorded on Varian inova-300 (300/75 MHz),inova-500 (500/125 MHz) and inova-600 (600/150 MHz) spectrometersequipped with sun workstations. Chemical shifts are reported in partsper million (ppm) relative to tetramethylsilane (TMS) as the internalstandard. NMR data is presented as follows: Chemical shift, multiplicity(s=singlet, d=doublet, t=triplet, dd=doublet of doublet, m=multipletand/or multiple resonances), coupling constant in Hertz (Hz),integration. All NMR signals were assigned on the basis of ¹H NMR, ¹³CNMR, COSY and HSQC experiments. Mass spectra were recorded on an AppliedBiosystems 5800 MALDI-TOF proteomics analyzer. The matrix used was2,5-dihydroxy-benzoic acid (DHB) and Ultramark 1621 as the internalstandard.

The synthesis ofβ-D-idopyranosyluronate)-(1→4)-(2-N-sulfoamino-2-deoxy-α/β-D-glucopyranoside)(7) (I0S0) is described below.

Benzyl(2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-β-D-glucopyranosyl)-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α/β-D-glucopyranoside(3)

Glycosyl donor 1 (467 mg, 1.05 mmol) and glycosyl acceptor 2 (877 mg,0.375 mmol) were combined in a flask, co-evaporated with toluene (3×3mL), and dissolved in DCM (8.7 mL). Powdered freshly activated 4 Åmolecular sieves were added, and the mixture was stirred for 30 min atambient temperature. The reaction mixture was cooled (0° C.) and NIS(0.236 g, 1.052 mmol) and TMSOTf (19.08 μL, 0.105 mmol) were added andthe stirring was continued until TLC indicated the consumption of donor(˜10 min). The mixture was then quenched with aqueous Na₂S₂O₃ andextracted with DCM (2×10 mL). The combined organic layers were dried(MgSO₄) and filtered and the filtrate was concentrated in vacuo. Theresidue was purified by silica gel chromatography using stepwisegradient of toluene and EtOAc (100-80%) to give disaccharide 3 (658 mg,73%). ¹H NMR (500 MHz, CDCl₃): δ 7.44-7.25 (m, 30H, CH Aromatic), 5.28(s, 2H, CH benzylidene×2), 5.00-4.96 (m, 4H, H2, H1α, H′2 CHH Bn), 4.92(d, 1H, J=11.5 Hz, CHH Bn), 4.84 (d, 1H, J=11.0 Hz, CHH Bn), 4.80 (d,1H, J=11.0 Hz, CHH Bn), 4.76-4.55 (m, 10H, CHH Bn×4, CHH Bn×2, CHH Bn×2,H6bα,β), 4.45 (dd, 1H, J=2.0 Hz, J=12.5 Hz, H6aα), 4.35 (d, 1H, J=8.0Hz, H1β), 4.14 (t, 1H, J=3.5 Hz, H5α), 4.11 (t, 1H, J=4.0 Hz, H6β),3.91-3.75 (m, 3H, H′5, H′4, H4β), 3.71 (bs, 1H, H′3), 3.51 (m, 1H, H2β),3.44-3.42 (m, 2H, H5β, H2a), 3.25 (t, 1H, J=9.5 Hz, H3β), 3.19 (d, 1H,J=11.0 Hz, H′6b), 3.10 (d, 1H, J=11.5 Hz, H′6a). ¹³C NMR (75.5 MHz,CDCl₃): δ 170.4, 170.2, 138.1, 137.9, 137.6, 128.9, 128.4, 128.3, 128.0,127.9, 127.9, 127.6, 127.4, 127.1, 126.1, 100.4, 98.0, 97.0, 81.2, 77.4,77.0, 76.5, 75.0, 74.9, 73.8, 73.7, 73.5, 72.1, 69.0, 69.0, 67.1, 62.3,60.3, 33.9, 24.8, 20.9, 20.8, 19.9, 19.8, 18.4, 18.3, −2.1, −3.3.HRMS-MALDI: (M+Na⁺) calcd for C₄₄H₄₇N₃O₁₂, 809.3159, found 809.3155.

Benzyl (methyl2-O-acetyl-3-O-benzyl-β-D-idopyranosyluronate)-(1→4)-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α/β-D-glucopyranoside)(4)

To a solution of disaccharide 3 (0.633 g, 0.781 mmol) in DCM was addedethanethiol (0.345 mL, 4.68 mmol) and p-TsOH (29.6 mg, 0.156 mmol). Theresulting reaction mixture was stirred at ambient temperature for 1 h.The reaction mixture was quenched with Et₃N and concentrated in vacuo.The residue was purified by silica gel column chromatography using astepwise gradient of toluene and EtOAc (100-80%) to give pure diol(0.543 g, 96%). To a vigorously stirred solution of the diol (0.533 g,0.738 mmol) in a mixture of DCM:H₂O (0.15 M, 2/1, v/v) was added TEMPO(22.96 mg, 0.147 mmol) and BAIB (0.594 g, 1.845 mmol). Stirring wascontinued until TLC indicated complete conversion of the startingmaterial to a spot of lower R_(f) (˜45 min). The reaction mixture wasquenched with aqueous Na₂S₂O₃ and the resulting mixture was extractedwith EtOAc (2×10 mL), and the combined organic layers were dried (MgSO₄)and filtered, and the filtrate was concentrated in vacuo. The oilyresidue was dissolved in THE (0.1 M) and treated with excess of freshlyprepared ethereal solution of diazomethane until the reaction mixturestayed yellow. The excess diazomethane was quenched by the addition ofAcOH until the mixture became colorless. The mixture was concentrated invacuo and the residue co-evaporated with toluene. The residue waspurified by silica gel column chromatography using stepwise gradient oftoluene EtOAc (100-50%) to give compound 4 (0.34 g, 83%, 2 steps). ¹HNMR (500 MHz, CDCl₃): δ 7.39-7.15 (m, 30H, CH Aromatic), 5.06 (bs, 2H,H′1), 4.98 (d, 1H, J=3.5 Hz, H1α), 4.92-4.88 (m, 3H, H′2, H′5, CHH Bn),4.74-4.58 (m, 7H, CHH Bn×4, CHH Bn×3), 4.50 (dd, 2H, J=1.5 Hz, 12.0 Hz,H6bα, H6bβ), 4.36 (d, 1H, J=12.5 Hz, H6aα), 4.31 (d, 1H, J=8.0 Hz, H1β),4.22-4.18 (m, 2H, H5α, H6aβ), 3.96 (bt, 1H, J=10.0 Hz, H′4), 3.88-3.85(m, 3H, H3α, H4α, H4β), 3.71 (d, 2H, J=2.5 Hz, H′3), 3.49-3.47 (m, 1H,H2β), 3.45 (s, 3H, CO₂CH₃), 3.41-3.36 (m, 2H, H2α, H5β), 3.25 (t, 1H,J=9.5 Hz, H3β), 2.10 (s, 3H, CH₃Ac), 2.06 (s, 3H, CH₃Ac). ¹³C NMR (75.5MHz, CDCl₃): δ 170.5, 170.5, 169.4, 169.3, 169.1, 137.8, 137.7, 137.1,136.4, 128.9, 128.5, 128.4, 128.1, 128.1, 128.0, 127.8, 127.4, 127.3,127.3, 125.2, 100.2, 97.9, 96.6, 81.1, 78.4, 77.4, 77.0, 76.6, 75.0,74.6, 74.4, 74.3, 74.3, 73.1, 72.2, 70.8, 69.8, 69.3, 68.4, 67.6, 67.1,67.0, 66.2, 63.4, 62.0, 51.9, 20.8. HRMS-MALDI: (M+Na⁺) calcd forC₃₄H₄₃N₃O₁₃, 749.2795, found 749.2790.

Benzyl(3-O-benzyl-β-D-idopyranosyluronate)-(1→4)-(2-azido-3-O-benzyl-2-deoxy-α/β-D-glucopyranoside)(5)

To a solution of compound 4 (70 mg, 0.09 mmol) in THF (1.4 mL) was addedLiOH (0.4 mL, 0.1 M) at room temperature. Stirring was continued for 40min, after which the reaction mixture was brought to pH 9.0 by theaddition of aqueous HCl (1.0 M), the resulting mixture was concentratedin vacuo and the residue was purified by column chromatography overlatrobeads using a stepwise gradient of toluene and methanol (100-70%).Appropriate fractions were concentrated in vacuo, to provide compound 5(60 mg, 99%). ¹H NMR (500 MHz, CD₃OD): δ 7.44-7.23 (m, 30H, Aromatic),5.04-4.98 (m, 4H, H′1, H1α, CHH Bn, CHH Bn), 4.94 (d, 2H, J=12.0 Hz, CHHBn), 4.92-4.75 (m, 3H, CHH Bn, CHH Bn, CHH Bn), 4.70 (d, 1H, J=12.0 Hz,CHH Bn), 4.63 (t, 2H, J=10.5 Hz, CHH Bn, CHH Bn), 4.58 (d, 1H, J=12.0Hz, CHH Bn), 4.44 (d, 1H, J=8.0 Hz, H1β), 4.41 (dd, 2H, J=4.5 Hz, J=19.0Hz, H6bα, H′5), 4.12 (t, 1H, J=9.5 Hz, H4a), 4.04 (t, 1H, J=9.5 Hz,H4β), 3.98-3.79 (m, 5H, H6a,bβ, H6aα, H5α, H3α), 3.57-3.52 (m, 4H,H′2×2, H′3×2), 3.46-3.43 (m, 2H, H3β, H5β), 3.39-3.35 (m, 2H, H2α, H2β).¹³C NMR (75.5 MHz, CD₃OD): δ 176.7, 140.2, 140.0, 138.7, 129.3, 129.2,129.0, 128.3, 101.9, 101.3, 101.2, 98.2, 82.5, 82.2, 82.0, 79.4, 77.5,77.0, 76.6, 76.0, 75.7, 75.0, 74.9, 73.8, 73.3, 73.0, 72.8, 72.4, 72.2,71.9, 70.5, 67.9, 64.3, 62.2, 62.1, 49.9, 49.7, 49.4, 49.1, 48.8, 48.5,48.2; HRMS-MALDI: (M+Na⁺) calcd for C₃₃H₃₇N₃O₁₁, 651.2428, found651.2422.

Benzyl(3-O-benzyl-β-D-idopyranosyluronate)-(1→4)-(2-N-sulfoamino-3-O-benzyl-2-deoxy-α-D-glucopyranoside)(6)

To a solution of compound 5 (20 mg, 0.03 mmol) in THF (2 mL) was added1.0 M solution of PM₃ in THF (0.24 mL, 0.24 mmol) and NaOH (0.1 M, 3.0mL, 0.3 mmol). The reaction mixture was stirred at room temperature for1 h and the progress of the reaction was followed by TLC(H₂O/acetonitrile, 10/90, v/v). The presence of the amino group wasindicated using ninhydrin as visualizing agent. After the completion ofthe reaction, pH was adjusted to 9.0 by careful addition of aqueous HCl(0.1 M). The mixture was concentrated in vacuo and the residueco-evaporated with toluene. To a solution of the crude residue inanhydrous methanol (5.0 mL) was added pyridinium sulfur trioxide (23.8mg, 0.15 mmol), and triethylamine (1.5 mL, 0.30 mmol) and sodiumhydroxide (0.6 mL, 0.06 mmol). Stirring was continued at roomtemperature for 1 h until RP-018 TLC (H₂O/methanol, 60/40, v/v)indicated the disappearance of the starting material. The mixture wasco-evaporated with toluene in vacuo and dissolved in H₂O and passedthrough a short column of Bio-Rad 50×8 Na⁺ resin (0.6×2.5 cm). Theresidue was applied to a small RP-018 column, which was eluted with astepwise gradient of water and methanol (90/10 to 60/40, v/v). Theappropriate fractions were lyophilized to give compound 6 (15 mg, 66%).¹H NMR (500 MHz, CD₃OD): δ 7.53-7.23 (m, 15H, CH Aromatic), 5.38 (d, 1H,J=3.5 Hz, H1α), 4.96 (d, 1H, J=4.5 Hz, H′1), 4.92 (d, 1H, J=10.5 Hz, CHHBn), 4.87-4.73 (m, 5H, CHH Bn, CHH Bn, CHH Bn, CHH Bn, CHH Bn), 4.59 (d,1H, J=11.0 Hz, CHH Bn), 4.46 (bs, 1H, H′5), 4.01-3.99 (M, 2H, H′4, H4a),3.85-3.83 (m, 2H, H6a,b), 3.74 (bd, 1H, J=9.5 Hz, H5α), 3.68 (t, 1H,J=10 Hz, H3α), 3.56 (m, 3H, H2α, H′2, H′3). ¹³C NMR (75.5 MHz, D20): δ176.8, 140.1, 139.53, 129.4, 129.4, 129.2, 129.1, 129.0, 128.5, 128.3,102.5, 100.5, 80.1, 79.9, 78.1, 74.6, 73.9, 73.5, 72.2, 71.7, 71.6,71.1, 63.4, 58.2, 49.8, 49.5, 49.3, 49.0, 48.7, 48.4, 48.1.

β-D-idopyranosyluronate)-(1→4)-(2-N-sulfoamino-2-deoxy-α/β-D-glucopyranoside)(7)

Pd/(OH)₂ on carbon (Degussa type, 20%, 1.5 times the weight of startingmaterial) was added to the solution of compound 6 (4 mg, 6 μmol) intBuOH and H₂O (1/1, v/v, 2 mL) and then placed under an atmosphere ofhydrogen. The reaction was completed after 16 h indicated by C18 TLC(H₂O/acetonitrile, 10/90, v/v). The mixture was filtered through Celiteand the filtrate was concentrated in vacuo. The residue was re-dissolvedin water and then passed through a short column of Bio-Rad 50×8 Na⁺resin (0.6×2.5 cm) using H₂O as eluent. Appropriate fractions werelyophilized to provide compound 7 (2 mg, 83%). ¹H NMR (500 MHz, D20),α-anomer: δ 5.45 (d, 1H, J=3.5 Hz, H1α), 4.81 (t, 2H, J=6.0 Hz, H′1),4.52 (t, 2H, J=4.5 Hz, H′5), 3.94-3.85 (m, 1H, H5α), 3.84-3.80 (m, 3H,H′4, H6a,b), 3.74-3.61 (m, 3H, H3α, H4α, H′3), 3.47-3.44 (m, 1H, H′2),3.26 (dd, 1H, J=3.5 Hz, J=10.0 Hz, H2α); ¹³C NMR (150 MHz, D20): δ176.6, 101.2, 91.2, 78.2, 72.7, 71.7, 71.3, 70.5, 69.6, 60.2, 58.2.

1-26. (canceled)
 27. A method of determining in an individual thepresence, identity, and/or severity of mucopolysaccharidosis (MPS) I,the method comprising: (a) generating a first biomarker comprising aglycan residual compound, wherein the first biomarker is generated bytreating a population of glycans, in or isolated from a biologicalsample from the individual, with at least one digesting glycan enzyme,wherein prior to enzyme treatment, the first biomarker is not present inabundance in samples from individuals with MPS I relative to individualswithout MPS I, and wherein the first biomarker is a non-reducing end(NRE) biomarker; (b) generating a second biomarker comprising a glycanresidual compound, wherein the second biomarker is generated by treatinga population of glycans, in or isolated from a biological sample fromthe individual, with at least one digesting glycan enzyme, wherein priorto enzyme treatment, the second biomarker is not present in abundance insamples from individuals with the MPS I relative to individuals withoutthe MPS I, and wherein: 1) the second biomarker is an NRE biomarkerdifferent from the first biomarker, 2) the second biomarker is areducing end biomarker, 3) the second biomarker is an internal glycanbiomarker, or 4) when the MPS I is caused by an abnormal function of aglycan degradation enzyme in the individual, the second biomarker isgenerated by treating the first biomarker with the glycan degradationenzyme that is functioning abnormally in the individual; (c) detectingthe presence of and/or measuring the amount of the first and secondbiomarker produced and displaying or recording the presence of or ameasure of a population of the first and second biomarkers by using ananalytical instrument; and (d) monitoring and/or comparing the amountsof the first and second biomarkers in a biological sample; wherein thepresence of and/or measure of the amounts of the first and secondbiomarkers are utilized to determine the presence, identity, and/orseverity of MPS I.
 28. The method of claim 27, wherein the population ofglycans comprises a glycan selected from the group consisting ofchondroitin sulfate, dermatan sulfate, and heparan sulfate.
 29. Themethod of claim 27, wherein the first biomarker is selected from thegroup consisting of IdoA-GlcNS, IdoA-GlcNS6S, IdoA-GlcNAc, andIdoA-GlcNAc6S.
 30. The method of claim 29, wherein the first biomarkeris IdoA-GlcNS.
 31. The method of claim 27, wherein the second biomarkeris selected from the group consisting of ΔUA-GlcN, ΔUA-GlcN6S,ΔUA2S-GlcN, AUAS-GlcN6S, ΔUA-GlcNAc, ΔUA-GlcNAc6S, ΔUA2S-GlcNAc,ΔUA2S-GlcNAc6S, ΔUA-GlcNS, ΔUA-GlcNS6S, ΔUA-GlcNS3S, ΔUA2S-GlcNS,ΔUA2S-GlcNS6S, ΔUA2S-GlcNS3S, ΔUA-GlcNS6S3S, and ΔUA2S-GlcNS6S3S. 32.The method of claim 31, wherein the second biomarker is selected fromthe group consisting of ΔUA-GlcNAc and ΔUA-GlcNS.
 33. The method ofclaim 27, wherein the first biomarker is selected from the groupconsisting of IdoA-GlcNS, IdoA-GlcNS6S, IdoA-GlcNAc, and IdoA-GlcNAc6S,and the second biomarker is selected from the group consisting ofΔUA-GlcN, ΔUA-GlcN6S, ΔUA2S-GlcN, AUAS-GlcN6S, ΔUA-GlcNAc, ΔUA-GlcNAc6S,ΔUA2S-GlcNAc, ΔUA2S-GlcNAc6S, ΔUA-GlcNS, ΔUA-GlcNS6S, ΔUA-GlcNS3S,ΔUA2S-GlcNS, ΔUA2S-GlcNS6S, ΔUA2S-GlcNS3S, ΔUA-GlcNS6S3S, andΔUA2S-GlcNS6S3S.
 34. The method of claim 33, wherein the first biomarkeris IdoA-GlcNS.
 35. The method of claim 33, wherein the second biomarkeris selected from the group consisting of ΔUA-GlcNAc, and ΔUA-GlcNS. 36.The method of claim 33, wherein the first biomarker is IdoA-GlcNS andthe second biomarker is selected from the group consisting of ΔUA-GlcNAcand ΔUA-GlcNS.
 37. The method of claim 27, wherein the first biomarkeris selected from the group consisting of IdoA-GalNAc4S, IdoA-GalNAc6S,IdoA-GalNAc, and IdoA-GalNAc4S6S, and the second biomarker is selectedfrom the group consisting of ΔUA-GalNAc, ΔUA-GalNAc4S, ΔUA-GalNAc6S,ΔUA2S-GalNAc, ΔUA2S-GalNAc4S, ΔUA2S-GalNAc6S, ΔUA-GalNAc4S6S, andΔUA2S-GalNAc4S6S.
 38. The method of claim 37, wherein the firstbiomarker is IdoA-GalNAc and the second biomarker is selected from thegroup consisting of ΔUA-GalNAc and ΔUA-GalNAc4S.
 39. The method of claim27, wherein the first biomarker is selected from the group consisting ofIdoA-GlcNS, IdoA-GlcNS6S, IdoA-GlcNAc, and IdoA-GlcNAc6S, and the secondbiomarker is selected from the group consisting of ΔUA-GalNAc,ΔUA-GalNAc4S, ΔUA-GalNAc6S, ΔUA2S-GalNAc, ΔUA2S-GalNAc4S,ΔUA2S-GalNAc6S, ΔUA-GalNAc4S6S, and ΔUA2S-GalNAc4S6S.
 40. The method ofclaim 39, wherein the first biomarker is IdoA-GlcNS.
 41. The method ofclaim 39, wherein the first biomarker is IdoA-GlcNS, and the secondbiomarker is selected from the group consisting of ΔUA-GalNAc andΔUA-GalNAc4S.
 42. The method of claim 27, wherein the first biomarker isselected from the group consisting of IdoA-GalNAc4S, IdoA-GalNAc6S,IdoA-GalNAc, and IdoA-GalNAc4S6S, and the second biomarker is selectedfrom the group consisting of ΔUA-GlcN, ΔUA-GlcN6S, ΔUA2S-GlcN,AUAS-GlcN6S, ΔUA-GlcNAc, ΔUA-GlcNAc6S, ΔUA2S-GlcNAc, ΔUA2S-GlcNAc6S,ΔUA-GlcNS, ΔUA-GlcNS6S, ΔUA-GlcNS3S, ΔUA2S-GlcNS, ΔUA2S-GlcNS6S,ΔUA2S-GlcNS3S, ΔUA-GlcNS6S3S, and ΔUA2S-GlcNS6S3S.
 43. The method ofclaim 42, wherein the first biomarker is IdoA-GalNAc and the secondbiomarker is selected from the group consisting of ΔUA-GlcNAc, andΔUA-GlcNS.
 44. The method of claim 27, wherein the first biomarker isselected from the group consisting of IdoA-GalNAc4S, IdoA-GalNAc6S,IdoA-GalNAc, and IdoA-GalNAc4S6S, and the second biomarker is selectedfrom the group consisting of IdoA-GlcNS, IdoA-GlcNS6S, IdoA-GlcNAc, andIdoA-GlcNAc6S.
 45. The method of claim 27, wherein the first biomarkeris selected from the group consisting of IdoA-GlcNS, IdoA-GlcNS6S,IdoA-GlcNAc, and IdoA-GlcNAc6S, and the second biomarker is selectedfrom the group consisting of IdoA-GalNAc4S, IdoA-GalNAc6S, IdoA-GalNAc,and IdoA-GalNAc4S6S.
 46. The method of claim 27, wherein the at leastone digesting glycan enzyme is iduronidase.
 47. A method of determiningresponse to a therapy in an individual having mucopolysaccharidosis(MPS) I, the method comprising: (a) generating a first biomarkercomprising a glycan residual compound, wherein the first biomarker isgenerated by treating a population of glycans, in or isolated from abiological sample from the individual, with at least one digestingglycan enzyme, wherein prior to enzyme treatment, the first biomarker isnot present in abundance in samples from individuals with MPS I relativeto individuals without MPS I, wherein the first biomarker is anon-reducing end (NRE) biomarker, (b) generating a second biomarkercomprising a glycan residual compound, wherein the second biomarker isgenerated by treating a population of glycans, in or isolated from abiological sample from the individual, with at least one digestingglycan enzyme, wherein prior to enzyme treatment, the second biomarkeris not present in abundance in samples from individuals with the MPS Irelative to individuals without the MPS I, and wherein: 1) the secondbiomarker is an NRE biomarker different from the first biomarker, 2) thesecond biomarker is a reducing end biomarker, 3) the second biomarker isan internal glycan biomarker, or 4) when the MPS I is caused by anabnormal function of a glycan degradation enzyme in the individual, thesecond biomarker is a biomarker generated by treating the firstbiomarker with the glycan degradation enzyme that is functioningabnormally in the individual, (c) detecting the presence of and/ormeasure the amount of the first and second biomarker produced anddisplaying or recording the presence of or a measure of a population ofthe first and second biomarkers by using an analytical instrument, and(d) monitoring and/or comparing the amounts of the first biomarker andthe second biomarker in a biological sample; and wherein the presence ofand/or measure of the amounts of the first and second biomarkers areutilized to monitor the treatment of the MPS I.
 48. The method of claim47, wherein the first biomarker is selected from the group consisting ofIdoA-GlcNS, IdoA-GlcNS6S, IdoA-GlcNAc, and IdoA-GlcNAc6S, and the secondbiomarker is selected from the group consisting of ΔUA-GlcN, ΔUA-GlcN6S,ΔUA2S-GlcN, AUAS-GlcN6S, ΔUA-GlcNAc, ΔUA-GlcNAc6S, ΔUA2S-GlcNAc,ΔUA2S-GlcNAc6S, ΔUA-GlcNS, ΔUA-GlcNS6S, ΔUA-GlcNS3S, ΔUA2S-GlcNS,ΔUA2S-GlcNS6S, ΔUA2S-GlcNS3S, ΔUA-GlcNS6S3S, and ΔUA2S-GlcNS6S3S. 49.The method of claim 47, wherein the first biomarker is IdoA-GlcNS andthe second biomarker is selected from the group consisting of ΔUA-GlcNAcand ΔUA-GlcNS.